Detection of Intratumoral Immune Cell Antigens in an Immunocompetent Glioblastoma Stem-like Cell Model Using Substrate-Based Immunohistochemistry​

Summary

The study provides a detailed, reproducible substrate-based immunohistochemistry workflow for consistent detection of single and dual antigens in formalin-fixed paraffin-embedded (FFPE) glioblastoma stem cell-derived tumor tissue sections. The protocol supports the identification of various immune and tumor markers while addressing technical challenges, with potential use in other tumor models.

Abstract

Glioblastoma (GBM) is the most common and lethal primary brain malignancy, lacking effective therapies. It is characterized by a profoundly immunosuppressive tumor microenvironment, driven in part by treatment-resistant GBM stem-like cells (GSCs). Here, we describe reproducible substrate-based single- and dual-color immunohistochemistry (IHC) protocols for detecting tumor-infiltrating immune cell-associated surface and intracellular antigens in formalin-fixed, paraffin-embedded (FFPE) brain tumor sections. The brain tumors were derived from mouse GSCs, which were orthotopically implanted in immunocompetent mice. The single-color IHC workflow uses a horseradish peroxidase (HRP)-based chromogenic detection system for visualization of individual immune cell antigens, whereas the dual-color IHC workflow employs a sequential alkaline phosphatase (AP)-based staining strategy for simultaneous detection of two immune cell antigens within the same tissue section, including workflows that use the same host origin primary antibodies. These methods provide a practical and reproducible framework for chromogenic analysis of tumor-infiltrating immune cell-associated antigens in GBM tissues and may be adaptable to other preclinical tumor models and potentially to human FFPE specimens.

Introduction

Glioblastoma (GBM) is an aggressive, highly invasive, and heterogeneous primary malignant brain tumor associated with poor patient survival despite current standard-of-care therapies¹. A defining feature of GBM is its profoundly immunosuppressive tumor microenvironment (TME), which contributes to resistance to immunotherapeutic strategies^2,3. Multiple mechanisms drive immunosuppression in GBM, including reduced infiltration of effector T cells and induction of T-cell dysfunction and exhaustion⁴. In addition, GBM stem-like cells (GSCs), a subpopulation of GBM cells, further promote immune evasion by maintaining an immunologically “cold” TME characterized by limited cytotoxic immune activity⁵. These features highlight the importance of accurately characterizing tumor-infiltrating immune cell populations and their functional states in the GBM TME.

Immunohistochemistry (IHC) remains one of the most widely used methods for evaluating immune cell infiltration within tumor tissues because it preserves tissue architecture while enabling visualization, localization, and semiquantitative assessment of cellular markers in situ^{6,7,8,9,10,11,12}. Previous immunohistochemical and immunoprofiling studies substantially advanced our understanding of the GBM immune landscape and identified immune cell populations associated with tumor progression and therapeutic response^{12,13,14,15,16}. Single-color chromogenic IHC is particularly useful for detecting individual immune markers in formalin-fixed, paraffin-embedded (FFPE) tissue sections^{12,13,15,17,18,19,20}. In contrast, dual-color chromogenic IHC enables simultaneous visualization of two markers within the same tissue section, facilitating assessment of cellular phenotype, proliferation, or activation state^12,19,21.

Despite these advances, practical and standardized substrate-based chromogenic IHC protocols applicable to immunocompetent GSC-derived GBM models remain limited. Moreover, chromogenic IHC remains more challenging in GBM than in many other solid tumors because of sparse and heterogeneous immune cell infiltration, extensive intratumoral heterogeneity, and the highly immunosuppressive and complex TME, which complicate the accurate visualization and interpretation of multiple cellular populations within the same tissue section^2,3,4. Dual chromogenic IHC presents additional technical challenges, particularly when both primary antibodies are raised in the same host species, requiring careful optimization of antibody compatibility, a sequential staining strategy for simultaneous detection of two antigens within the same tissue section, and selection of a chromogenic substrate to ensure clear marker discrimination under light microscopy.

Here, we describe reproducible substrate-based single- and dual-color IHC protocols that successfully detect representative tumor-infiltrating immune cell-associated surface and intracellular antigens in an immunocompetent orthotopic 005 GSC-derived GBM model^22,23,24. These substrate-based chromogenic IHC protocols can also be adapted to other preclinical tumor models and potentially to human FFPE tumor specimens. The harvested mouse brain tissues were fixed in 10% neutral buffered formalin overnight (~12 h) before routine tissue processing and paraffin embedding, as previously described¹⁹. FFPE blocks were sectioned (5 µm thickness¹⁹) onto positively charged microscope glass slides and stored at room temperature until use. Single-color IHC uses a horseradish peroxidase (HRP)-based chromogenic detection system with 3,3′-diaminobenzidine (DAB) as the substrate to generate a brown signal for visualization of individual antigens under light microscopy. This workflow is suitable for detecting both membrane-associated and intracellular antigens in FFPE tissue sections.

Dual-color IHC enables simultaneous detection of two antigens within the same tissue section but requires additional optimization to ensure antibody compatibility and clear chromogenic signal separation. Dual-color IHC staining is relatively straightforward when the primary antibodies are raised in different host species, allowing the use of species-specific secondary antibodies. However, simultaneous detection becomes more technically challenging when both primary antibodies originate from the same host species. To address this challenge, the dual-color IHC workflow described here employs a sequential alkaline phosphatase (AP)-based chromogenic staining strategy using distinct substrates to permit clear visual discrimination between two antigen signals under light microscopy.

Both workflows include tissue deparaffinization, rehydration, antigen retrieval, blocking, staining, dehydration, clearing, and mounting steps. Preparation of reagents is crucial for staining quality and reproducibility (see Supplementary File 1). Likewise, appropriate dilutions of primary antibodies are also critical for a successful staining protocol, which is done immediately before use in 2.5% normal horse or goat serum, depending on the host species of the corresponding secondary antibody (see Table 1). A list of species-specific secondary antibodies used for single and dual-color staining is also presented in Table 2. Single-color IHC uses xylene-based clearing and mounting reagents, whereas dual-color IHC requires xylene-free alternatives (e.g., Histo-Clear), as the substrate is partially soluble in xylene (see the list of substrates in Table 3).

Protocol

This protocol was conducted in accordance with all relevant regulatory and institutional guidelines, including the Institutional Animal Care and Use Committee (IACUC) (IACUC protocol numbers: 2004N000067 and LA24-0045).

NOTE: Several steps in both single- and dual-color IHC protocols involve flammable reagents, including ethanol and tissue-clearing agents. Accordingly, procedures involving flammable chemicals, including deparaffinization, rehydration, dehydration, clearing, and mounting, should be performed in a certified chemical fume hood using appropriate laboratory safety practices. Flammable reagents should be prepared and stored in properly labeled, screw-capped glass containers within designated flammable storage cabinets, in accordance with institutional safety guidelines. The materials and reagent volumes are configured for batch processing of up to 48 microscope slides (two staining racks, 24 slides per rack), although the workflow can be readily scaled according to experimental needs.

1. Single-color IHC for FFPE brain tumor sections

NOTE: The following protocol describes substrate-based single-color IHC for the detection of representative surface and intracellular antigens in FFPE brain tumor sections derived from the orthotopic 005 GSC model.

1. Deparaffinization (20 min):
   1. Put slides in a polyoxymethylene (POM) slide rack and immerse the slide rack in staining containers, first in Xylene I and then in Xylene II, each for 10 min in a fume hood. Ensure tissue sections remain fully submerged in xylene during deparaffinization.
      CAUTION: Perform this step under a chemical fume hood with appropriate personal protective equipment (PPE), including a laboratory coat, eye goggles, a face mask, and hand gloves. Do not allow sections to dry at this stage or any of the following steps, as this can cause tissue damage and section loss. Store xylene solutions in airtight glass containers and reuse them if they appear clean.
2. Gradual rehydration (15 min):
   1. Rehydrate sections sequentially in 100%, 90%, and 70% ethanol for 5 min each. For this, immerse slides in 100% ethanol in the staining container for 5 min (then return the 100% ethanol to the glass bottle for reuse), followed by 90% ethanol for 5 min (then return it to the bottle), and finally in 70% ethanol for 5 min (then return it to the bottle).
      CAUTION: Perform this step under a chemical fume hood and use PPE as in step 1.1. These ethanol solutions can be reused across experiments if stored in airtight glass containers, provided they appear clean.
3. Water rinse (5 min):
   1. Place slides in distilled water for 5 min, and this completes the rehydration process.
      NOTE: While running this step, prepare 600 mL (for 1 slide rack) to 800 mL (for 2 slide racks) of 1× antigen retrieval buffer solution (10 mM sodium citrate buffer) in a 2 L microwaveable glass beaker, as per the instructions listed in Supplementary File 1.
4. Antigen retrieval (~20 min):
   1. Cover the 2 L beaker containing the freshly prepared 10 mM sodium citrate antigen retrieval buffer with plastic wrap to prevent excessive evaporation during pre-heating and prolonged microwave heating.
   2. Make a few holes in the plastic wrap to prevent the buffer from blowing off due to excess pressure generated during boiling the tissue sections.
      NOTE: Use PPE as in step 1.1.
   3. Preheat the freshly prepared 10 mM sodium citrate antigen retrieval buffer solution for 3 min. Place the slide rack with rehydrated tissue sections into the prewarmed antigen retrieval solution.
   4. Then microwave tissue sections for 15 min at the highest microwave power (e.g., P10), carefully monitoring the beaker to ensure the sections remain submerged and do not dry out due to evaporation.
      NOTE: Alternatively, perform antigen retrieval in a plastic staining trough placed inside a steam cooker for approximately 15 min, which provides a gentler heating method with less monitoring. Sodium citrate buffer (10 mM, pH 6.0) was successfully validated and used as an antigen retrieval buffer for all primary antibodies listed in Table 1. Depending on the primary antibody and the manufacturer's recommendations, other buffers, such as EDTA or Tris-EDTA, may be required for antibodies not evaluated in this study. 
5. Cooling (10–20 min):
   1. Remove the beaker from the microwave, place it on the working bench, and allow it to cool to room temperature for 20 min. To accelerate cooling, place the beaker in ice-cold tap water for ~10 min.
      CAUTION: It will be extremely hot; use appropriate heat-resistant gloves or beaker tongs to handle it. Use PPE as in step 1.1.
6. Dulbecco's phosphate-buffered saline (DPBS) wash (~10 min):
   1. Remove the slide rack from the beaker and immerse slides in 1× DPBS solution in the staining container.
   2. Rinse sections twice in 1× DPBS for 5 min each using gentle, static washes to avoid tissue detachment.
7. Hydrophobic Barrier (<1 min/slide):
   1. During the DPBS wash (step 1.6), carefully remove each slide individually, gently wipe around the tissue section with lint-free wipes (e.g., Kim Wipes), avoiding contact with the tissue itself.
   2. Use a hydrophobic barrier, such as a peroxidase-anti-peroxidase (PAP) pen, to draw a liquid-repellent barrier around each tissue section, then return the slide to the DPBS wash solution in the staining container. Repeat these steps for all slides.
      NOTE: A hydrophobic barrier around the tissue prevents reagent spillover between sections and helps retain antibodies on the tissue.
8. Block endogenous peroxidase (5 min):
   1. Discard 1× DPBS from the staining container, then add 3% H₂O₂ solution, ensuring the slides or tissue sections are fully immersed for 5 min.
      NOTE: Prolonged incubation in 3% H₂O₂ may damage tissue. Blocking endogenous peroxidase activity is a crucial step to prevent background caused by endogenous peroxidase during color development. Use PPE as in step 1.1.
9. DPBS wash (~12 min):
   1. Discard the 3% H₂O₂ solution, and rinse slides again in 1× DPBS twice for 5 min each.
   2. After the second wash, remove the slides from the slide rack, place them flat in the humidified chamber, and add distilled water to the edges of the humidified chamber.
   3. Do not let the sections dry, so proceed immediately to blocking in step 1.10.
      NOTE: Each humidified chamber listed in the Table of Materials has space for and can accommodate 30 slides.
10. Serum blocking (~70 min):
    1. Add 50 µL of 5% bovine serum albumin (BSA) solution per section and incubate for 30 min at room temperature while keeping the humidified chamber lid on to prevent evaporation of the blocking buffer from the tissue sections.
    2. After 30 min, gently tap the slides on lint-free wipes placed on the workbench to discard the BSA.
    3. Then, add 2.5% normal horse or goat serum (one drop per section), matching the host species of the secondary antibody, and incubate the sections for an additional 30 min at room temperature with the humidified chamber lid on, gently tap the slides on absorbent paper or lint-free wipes to remove the serum, and proceed immediately to step 1.11.
       NOTE: Do not let the sections dry out. Prepare the primary antibodies diluted in the appropriate blocking buffer (see Table 1) near the end of the blocking step so they are ready for use in step 1.11 once blocking is complete.
11. Primary antibody incubation (~16 h):
    1. Apply ~50 µL of primary antibodies, optimally diluted in the appropriate blocking buffer (see Table 1), to tissue sections and incubate overnight (~16 h) at 4 °C in a humidified chamber.
       NOTE: Ensure the designated areas of the humidified chamber are filled with distilled water to keep the chamber humidified overnight. Additionally, take precautions to ensure that the designated areas/chambers for tissue slides are not filled with distilled water, as this can cause flooding of the tissue sections.
12. DPBS (0.1% Tween 20) wash (~18–20 min):
    1. Place a slide rack in a staining container and fill it with wash buffer (1× DPBS, 0.1% Tween 20).
    2. After overnight incubation, remove the slides from the humidified chamber, discard the primary antibodies by gently tapping the slides on absorbent paper, then place the slides in a slide rack immersed in wash buffer (1× DPBS, 0.1% Tween 20) in a staining container, and discard the wash buffer.
    3. Then, wash sections in 1× DPBS (0.1% Tween 20) three times, 5 min each, and then, remove the slides from the slide rack and place them flat in the humidified chamber, and proceed immediately to step 1.13.
       NOTE: Each wash time can be extended to 10–15 min if background staining appears to be observed high after color development in step 1.15. Washing should be gentle and static (no rocking or shaking) to prevent tissue detachment from the slides.
13. Secondary antibody incubation (~32–35 min):
    1. Add one drop/section of the appropriate HRP-conjugated secondary antibody (anti-rabbit or anti-rat IgG, as appropriate; see Table 2) and incubate the tissue sections for 30 min at room temperature with the humidified chamber lid on.
       NOTE: The secondary antibody can be diluted 1:1 in PBS to reduce background in step 1.15.
14. DPBS (0.1% Tween 20) wash (~18–20 min):
    1. After incubation with the secondary antibody, remove the slides from the humidified chamber and gently tap the slides on absorbent paper to discard the secondary antibodies.
    2. Then, place the slides in a slide rack immersed in wash buffer (1× DPBS with 0.1% Tween 20) inside a staining container for a brief wash, and discard the wash buffer.
    3. Then, wash tissue sections three times with 1× DPBS (0.1% Tween 20) for 5 min each.
       1. Before starting the chromogen development step (i.e., step 1.15), place a separate slide rack in a staining container filled with distilled water, a preparatory step for step 1.16, to stop the chromogenic reaction.
       2. During the final wash, freshly prepare the DAB working solution (1 drop of chromogen per 1 mL of substrate; see Supplementary File 1).
15. Chromogen development (30 s–5 min):
    1. After the third wash in step 1.14, remove the slides from the slide rack and lay them flat on the workbench with a white background.
    2. Apply 50 µL of DAB working solution to each section and visually monitor the development of a brown color against a white background or under a light microscope.
       NOTE: Use PPE as in step 1.1. Using white absorbent paper helps visualize brown color development during the DAB chromogen stage. Color development may take 20–60 s for some markers and 2–5 min for others (see details in step 1.16).
16. Stop reaction (1–2 min):
    1. As soon as the desired brown color signal appears in step 1.15, immediately stop the reaction by dipping the slide into a container of distilled water prepared in step 1.14.3.1.
    2. Keep slides rinsed in distilled water for 1 min. Discard the distilled water, then proceed to step 1.17 (counterstaining).
       NOTE: Slides can remain in distilled water for up to 1 h without affecting staining. The time required for color development dictates when to initiate the stop reaction step. Additionally, if there are 2–3 markers (such as CD3, CD4, CD8, or others) on the same slide (as is the case in this study), reaction times may vary; for example, in the system described here, color development generally takes around 30 s for CD8⁺ cells, 1–3 min for CD3⁺ or CD4⁺ cells, or longer (3–5 min) for Granzyme B⁺ and Ki67⁺ cells. Due to differences in the timing of color development, the entire slide cannot be immersed in distilled water to stop the reaction. Instead, stop each reaction individually for each section by adding 200–300 µL of distilled water directly onto each section.
17. Counterstaining (<1 min):
    1. To enhance contrast with brown staining, add diluted hematoxylin (1:3 in distilled water) to the staining container, ensuring that the slides remain immersed in diluted hematoxylin for ~10–20 s.
       NOTE: If the DAB background is strong, counterstain longer (up to 1 min) or use undiluted hematoxylin briefly.
18. Tap water wash (6 min):
    1. Discard the hematoxylin from the staining container and return it to the glass bottle for reuse.
    2. Rinse the slides in tap water for 1 min, then rinse in slow-running tap water for 5 min, keeping sections facing away from the direct flow of water. Discard tap water and proceed to step 1.19.
       NOTE: The remaining steps until mounting are performed in a fume hood.
19. Gradual dehydration (15–17 min):
    1. Immerse slides in 70% ethanol in the staining container for 5 min (then return the 70% ethanol to the glass bottle for reuse), followed by 90% ethanol for 5 min (then return it to the bottle), and finally in 100% ethanol for 5 min (then return it to the bottle).
    2. Proceed to step 1.20.
       NOTE: Perform this step in a chemical fume hood. The same ethanol solutions from step 1.2 are used in this dehydration step and can be reused many times if they appear clean.
20. Clearing:
    1. Use the same Xylene I and II solutions used for deparaffinization (step 1.1) to clean the tissue sections/slides.
    2. First, immerse slides in Xylene I solution in the staining container for 10 min (then return the Xylene I to the glass bottle for reuse) and then in Xylene II solution for an additional 10 min.
    3. Keep slides in Xylene II until mounting (i.e., step 1.21) is complete.
       NOTE: Never let sections dry during this step, as dried sections become fragile; thus, if needed, sections may remain in Xylene II longer for mounting.
21. Mounting:
    1. Remove slides one by one from Xylene II and place them flat in a chemical fume hood. Place one drop of Xylene-based mounting medium on an appropriately sized coverslip, then place the coverslip on the slide, with the mounting medium facing the tissue sections.
    2. Gently press the coverslip with the thumb to spread the mounting medium evenly across all three sections and to remove air bubbles.
       NOTE: 24 × 50 mm coverslip is required to cover three sections on a slide; 24 × 40 mm coverslip for two sections; and 24 × 24 mm coverslip for one section. It is important to complete mounting within 30 s per slide because the mounting medium can dry quickly due to the constant airflow in the fume hood, causing Xylene II to quickly evaporate from the slide and making the sections fragile.
22. Air drying and light microscopy:
    1. Air-dry slides in the fume hood for at least 2 h before evaluation and imaging using a light microscope. Once dried, store the mounted slides in slide boxes at room temperature for years.

2. Dual-color IHC for FFPE brain tumor sections

NOTE: The following protocol describes a substrate-based dual-color chromogenic IHC workflow for simultaneous detection of two antigens within the same FFPE brain tumor section. Compared with single-color IHC, the dual-color workflow is more time-intensive because it requires sequential staining cycles with independent primary antibody incubations, including overnight incubation steps for each staining round. However, this extended workflow enables reliable simultaneous antigen detection within the same tissue section while preserving tissue morphology and maintaining clear chromogenic signal separation under light microscopy.

1. Deparaffinization (20 min):
   1. Follow the same deparaffinization process as outlined above for single-color IHC in step 1.1 in a chemical fume hood, but use the non-xylene-based clearing agents (e.g., Histo-Clear I and Histo-Clear II instead of xylene).
      CAUTION: Use PPE as in step 1.1 and perform this step under a chemical fume hood. Histo-Clear is recommended because the chromogen is partially soluble in xylene, and xylene-free processing helps preserve staining intensity during dual-color IHC. Do not allow sections to dry. These clearing solutions can be reused if stored in airtight glass containers and appear clean.
2. Gradual rehydration (15 min):
   1. Same as single-color IHC as outlined in step 1.2. Rehydrate sections sequentially with a gradual decrease in ethanol concentration (100%, 90%, and 70%) for 5 min in each step.
      ​NOTE: A separate set of xylene-free graded ethanol is used here since the graded ethanol used for single-color IHC in step 1.2 is likely contaminated with Xylene II.
3. Water rinse (5 min):
   1. Same as single-color IHC in step 1.3; place slides in distilled water for 5 min to complete the rehydration process.
      ​NOTE: Same as step 1.3; prepare antigen retrieval solution during this 5-min period.
4. Antigen retrieval (~20 min): Follow the same antigen retrieval procedure as outlined for single-color IHC in step 1.4.
5. Cooling (10–20 min): Follow the same cooling process as described for single-color IHC in step 1.5.
6. DPBS wash (10 min): Follow the process of washing off the antigen retrieval solution as outlined for single-color IHC in step 1.6.
7. Hydrophobic barrier (<1 min/slide):
   1. During the DPBS wash in step 2.6, make a hydrophobic barrier around the tissue sections following the same process/protocol as in step 1.7.
   2. Use the hydrophobic barrier pen to draw a liquid-repellent barrier around each tissue section, then return the slide to the DPBS wash solution in the staining container. Repeat these steps for all slides.
8. Block alkaline phosphatase (5 min):
   1. After completing the hydrophobic barriers on all tissue sections, remove the slides from the staining container, place them flat in the humidified chamber, add one drop of endogenous alkaline phosphatase blocking solution (e.g., BLOXALL) per section, and incubate sections for 5 min. Proceed immediately to step 2.9.
      ​NOTE: Since AP-conjugated secondary antibodies are used in the dual-color IHC protocol, it is essential to block endogenous alkaline phosphatase activity to reduce background signal during red or blue color development. BLOXALL blocking solution is used here to inhibit endogenous alkaline phosphatase. It also inhibits endogenous peroxidase, so this blocking solution can also be used in the single-color IHC protocol to block endogenous peroxidase activity, instead of 3% H₂O₂ solution.
9. DPBS wash (10 min):
   1. Rinse slides in 1× DPBS solution in the staining container twice for 5 min each.
   2. After the second wash, remove the slides from the staining container, place them flat in the humidified chamber, and add distilled water to the edges of the humidified chamber.
      NOTE: Do not let the sections dry, so proceed immediately to blocking non-specific antigen-antibody binding in step 2.10.
10. Serum blocking (~70 min):
    1. Follow the same serum blocking process as in the single-color IHC protocol outlined in step 1.10.
       1. First, block with the universal blocking buffer (5% BSA; 50 µL/section) for 30 min at room temperature and then with the 2.5% normal horse serum (one drop/section), matching the host species of the secondary AP-conjugated anti-rabbit IgG antibody, for another 30 min at room temperature with the humidified chamber lid on.
       2. After this period, gently tap the slides on absorbent paper to discard the serum, and proceed to step 2.11.
          NOTE: Prepare primary antibody I (see Table 1) during the final min of the blocking step, as described in step 1.10.
11. Primary antibody I incubation (~16 h):
    1. Follow the same incubation process with the primary antibody as in step 1.11. For example, add ~50 µL of rabbit anti-CD3 antibody, diluted 1:100 in 2.5% normal horse serum (see Table 1), to each tissue section.
    2. Incubate overnight (~16 h) at 4 °C in a humidified chamber.
12. DPBS (0.1% Tween 20) wash (~18–20 min):
    1. Follow the same wash procedure (3 wash steps, 5 min each) as outlined for the single-color IHC protocol in step 1.12.
    2. After the third wash, place slides flat inside the humidified chamber. Proceed immediately to step 2.13.
       NOTE: Same notes as step 1.12, e.g., each wash time can be extended to 10–15 min, and washing should be gentle and static. Do not let the sections dry.
13. Secondary antibody I incubation (~32–35 min):
    1. Add one drop per section of the AP-conjugated anti-rabbit IgG secondary antibody (see Table 2) and incubate the tissue sections for 30 min at room temperature with the humidified chamber lid on.
       NOTE: The secondary antibody can be diluted 1:1 in PBS to reduce background in step 2.15.
14. DPBS (0.1% Tween 20) wash (~18–20 min):
    1. Follow the same wash procedure with 1× DPBS/0.1% Tween 20, as in step 1.14.
    2. NOTE: During the final wash step, freshly prepare the substrate working solution (5 mL, as recommended; see Table 3) according to the manufacturer’s instructions (see reagent preparation in Supplementary File 1).
15. Red color development (12–15 min):
    1. After completing step 2.14, place slides flat on the workbench with a white background, such as white absorbent paper, as described in step 1.15.
    2. Mix the freshly prepared red substrate working solution thoroughly, then add 50 µL to each tissue section (discard any leftover solution).
    3. Incubate the tissue sections at room temperature until the red color appears, checking periodically every 2–3 min against a white background or under light microscopy.
       NOTE: The time required for red color development varies among markers and can take 20–30 min. However, for staining of CD3⁺ cells in 005 brain tumor sections with the AP-detection system, red color development usually occurs within 12–15 min.
16. Buffer rinse (5 min):
    1. As soon as the desired red color signal appears in step 2.15, rinse sections in 1× DPBS/0.1% Tween 20 for 5 min in a staining container.
    2. During this wash time, freshly prepare the primary antibody II in an appropriate blocking buffer (e.g., rabbit anti-Ki67 antibody diluted 1:100 in 2.5% normal horse serum; see Table 1).
       NOTE: Unlike the distilled water rinse used in single-color IHC (step 1.16), DPBS/0.1% Tween 20 is used here not only to stop the reaction but also to rigorously wash away excess chromogen and prepare for the second staining cycle.
17. Primary antibody II incubation (~16 h):
    1. Bring slides into a humidified chamber and add ~50 µL of freshly prepared rabbit anti-Ki67 antibody to each tissue section. Incubate overnight (~16 h) at 4 °C.
18. DPBS (0.1% Tween 20) wash (~18–20 min):
    1. Follow the same procedure as step 1.12.
    2. After the third wash, remove the slides from the staining container and place them flat in the humidified chamber. Proceed immediately to step 2.19.
19. Secondary antibody II incubation (~32–35 min):
    1. Follow the same procedure as step 2.13. Add one drop per section of the AP-conjugated anti-rabbit IgG (see Table 2) and incubate the tissue sections for 30 min at room temperature with the humidified chamber lid on.
20. DPBS (0.1% Tween 20) wash (~18–20 min):
    1. Follow the same wash procedure with 1× DPBS/0.1% Tween 20, as in step 1.14.
       NOTE: During the final wash step, freshly prepare the substrate working solution (5 mL, as recommended; see Table 3) according to the manufacturer’s instructions (see reagent preparation in Supplementary File 1).
21. Blue color development (10–12 min):
    1. After completing step 2.20, place slides flat on the workbench with a white background.
    2. Mix the freshly prepared blue substrate working solution thoroughly, then add 50 µL to each tissue section (discard any leftover solution).
    3. Incubate the tissue sections at room temperature until the blue color appears, checking periodically every 2–3 min against a white background or under light microscopy.
       NOTE: Like the red color development in step 2.15, the time required for blue color development can take 20–30 min. However, for staining of Ki67⁺ cells in 005 brain tumor sections with the AP-detection system, blue color develops within 10–12 min.
22. Buffer wash (5 min):
    1. As soon as the desired blue color signal appears in step 2.21, immerse sections in 1× DPBS/0.1% Tween 20 for 5 min in a staining container. Discard the wash solution.
23. Water rinse (1 min): Rinse the sections in distilled water for 1 min. Discard distilled water after rinsing.
24. Gradual dehydration (15–17 min):
    1. Immerse slides in 70% ethanol in the staining container for 5 min (then return the 70% ethanol to the glass bottle for reuse), followed by 90% ethanol for 5 min (then return it to the bottle), and finally in 100% ethanol for 5 min (then return it to the bottle).
    2. Repeat each step in a fume hood. Proceed to step 2.25.
       NOTE: The same (xylene-free) ethanol solutions from step 2.2 are used in this dehydration step and can be reused many times if they appear clean.
25. Non-xylene-based clearing:
    1. Use the same xylene-free solutions (i.e., Histo-Clear) used in step 2.1 to clean the tissue sections/slides.
       1. First, immerse slides in xylene-free solution I in the staining container for 10 min (then return the xylene-free solution I to the glass bottle for reuse) and then in xylene-free solution II for an additional 10 min.
       2. Keep slides in xylene-free solution II until mounting (i.e., step 2.26) is complete.
          NOTE: Sections may remain in xylene-free solution II longer, if needed, for mounting.
          CAUTION: Use PPE as in step 1.1 and perform this step under a chemical fume hood.
26. Non-xylene-based mounting:
    1. Remove slides one by one from xylene-free solution II and place them flat in a fume hood. Place one drop of non-xylene-based mounting medium on an appropriately sized coverslip, then place the coverslip on the slide, with the mounting medium facing the tissue sections.
    2. Gently press the coverslip with the thumb to spread the mounting medium evenly across all three sections and to remove air bubbles.
       NOTE: Complete mounting within 30 s per slide, as the mounting medium can dry quickly. Use PPE as in step 1.1 and perform this step under a chemical fume hood.
27. Air drying and light microscopy: Air-dry slides in the fume hood for at least 2 h before evaluation and imaging using a light microscope. Once dried, the mounted slides can be stored in slide boxes at room temperature for years.

Results

Single-color IHC using the HRP-based chromogenic detection system produced clear and reproducible staining in FFPE 005 brain tumor sections. Representative images of surface antigen staining are shown in Figure 1, demonstrating successful detection of T-cell markers (CD3, CD4, CD8), tumor-associated macrophage (TAM) markers (CD68, F4/80), and the immune checkpoint molecule programmed death ligand 1 (PD-L1). In all cases, positive staining was visualized as a distinct brown chromogenic signal using DAB as the chromogen, with preserved tissue morphology and minimal background staining, indicating that the single-color IHC protocol is compatible with diverse membrane-associated antigens.

The applicability of the single-color IHC protocol for intracellular antigen detection is demonstrated in Figure 2. Representative staining images show successful detection of transcriptional, signaling, proliferative, cytotoxic, and apoptotic intracellular markers, including T-bet, FoxP3, phosphorylated STAT1 (pSTAT1), Ki67, granzyme B, and cleaved caspase-3. Similar to surface antigen staining, intracellular marker detection produced clear brown chromogenic signals while preserving tissue architecture, with minimal nonspecific background staining (Figure 2), demonstrating that the protocol is suitable for detecting both surface and intracellular antigens.

The dual-color IHC protocol was successfully used to simultaneously detect two antigens within the same tissue section, employing a sequential alkaline phosphatase (AP)-based chromogenic detection strategy. As shown in Figure 3, co-staining of CD3(red)/Ki67(blue) and pSTAT1(red)/CD68(blue), which are indicative of proliferative T cells and M1-like macrophages, respectively¹⁹, produced clearly distinguishable chromogenic signals under light microscopy, demonstrating effective simultaneous dual-antigen detection with clear color separation using the sequential staining workflow. To further demonstrate the versatility of this dual-color staining approach, the same protocol was successfully applied to co-detect a neuronal marker (NeuN in red) and green fluorescent protein (GFP in blue) in brain tumor sections derived from GFP-expressing 005 GSC tumors (Figure 4). Colocalization of GFP and NeuN likely indicates that 005 GSCs differentiate into mature phenotypes expressing the neuronal marker NeuN²¹.

The images in Figure 1, Figure 2, and Figure 3 are representative of four independent tumors, with one section per tumor for all stained immune cell antigens, except the F4/80 image, which represents two independent tumors, and the PD-L1 image, which represents three independent tumors, with two sections for both F4/80 and PD-L1. The image in Figure 4 represents a single tumor section. Because the dual-IHC protocol was used to sequentially stain two antigens (e.g., CD3⁺ and Ki67^+, or CD68⁺ and pSTAT1⁺) in the same tissue section across two to four independent tumors and was further validated by co-staining of GFP⁺ and NeuN⁺ cells, these results support the reproducible application of the dual-IHC protocol. Similarly, the single-IHC protocol was successfully applied to 12 independent immune cell antigens, demonstrating its broad applicability under the staining conditions described here.

Together, these representative results demonstrate that the described substrate-based single- and dual-color IHC protocols provide practical and reproducible methods for detecting a broad range of surface and intracellular antigens in FFPE GBM tissues.

Figure 1: Single-color immunohistochemistry (IHC) for surface antigens. Substrate-based single-color (brown) IHC staining of surface antigens for tumor-infiltrating T cells (CD3⁺, CD4⁺, and CD8⁺), tumor-associated macrophages (CD68⁺ and F4/80⁺), and immune checkpoint molecule programmed death ligand 1 (PD-L1⁺) using the horseradish peroxidase (HRP)-based detection system in glioblastoma (GBM) stem-like cell (GSC)-derived mouse GBM sections (see details in Section 1 of the protocol). Representative IHC images are presented (10× magnification for CD3, CD4, and CD8; 20× magnification for CD68, F4/80, and PD-L1). Positive cells are stained brown. Scale bars = 100 μμm. Please click here to view a larger version of this figure.

Figure 2: Single-color immunohistochemistry (IHC) for intracellular antigens. Substrate-based single-color (brown) IHC staining of intracellular transcriptional (T-bet⁺ and FoxP3⁺), signaling (pSTAT1⁺), proliferative (Ki67⁺), cytotoxic (granzyme B⁺, GranB), and apoptotic (cleaved caspase-3⁺, CC3) markers in brain tumor sections using the horseradish peroxidase (HRP)-based detection system in glioblastoma (GBM) stem-like cell (GSC)-derived mouse GBM sections (see details in Section 1 of the protocol). Representative IHC images are presented (10× magnification for FoxP3, GranB, and CC3; 20× magnification for T-bet, pSTAT1, and Ki67). Positive cells are stained brown. Scale bars = 100 μμm. Please click here to view a larger version of this figure.

Figure 3: Dual-color immunohistochemistry (IHC) for proliferative tumor-infiltrating lymphocytes (TILs) and M1-like tumor-associated macrophages (TAMs). Substrate-based dual-color (red/blue) IHC staining of CD3⁺(red)/Ki67⁺(blue) tumor-infiltrating proliferative T cells and pSTAT1⁺(red)/CD68⁺(blue) M1-like macrophages using the alkaline phosphatase (AP)-based detection system in glioblastoma (GBM) stem-like cell (GSC)-derived mouse GBM sections (see details in Section 2 of the protocol). Representative IHC images are presented (20× magnification). In the top image, CD3⁺ cells are stained red, Ki67⁺ cells are stained blue, and a few of the colocalized cells are indicated in black arrows. Similarly, in the bottom image, pSTAT1⁺ cells are stained red, CD68⁺ cells are stained blue, and a few of the co-localized cells are indicated with black arrows. Scale bars = 100 μμm. Please click here to view a larger version of this figure.

Figure 4: Dual-color immunohistochemistry (IHC) for neuronal cells and green fluorescent protein-positive (GFP⁺) glioblastoma stem-like cells (GSCs). Substrate-based dual-color (red/blue) IHC staining of NeuN⁺(red)/GFP⁺(blue) using the alkaline phosphatase (AP)-based detection system in glioblastoma (GBM) stem-like cell (GSC)-derived mouse GBM sections (see details in Section 2 of the protocol). Representative IHC image is shown (20× magnification). NeuN⁺ (a neuronal marker) cells are stained red, GFP⁺ (expressed by 005 GSCs) cells are stained blue, and a few of the colocalized cells are indicated in black arrows. Scale bar = 100 μμm. Please click here to view a larger version of this figure.

Primary Antibody Against	Catalog Number	Vendor	Working Dilution	Dilution Buffer**	Host Species	Appropriate Secondary
CD3e	ab5690	Abcam	1:100	Horse serum	Rabbit	Anti-rabbit IgG
CD4	14-9766-80	eBioscience	1:200	Goat serum	Rat	Anti-rat IgG
CD68	ab125212	Abcam	1:100	Horse serum	Rabbit	Anti-rabbit IgG
CD8a	14-0808-80	eBioscience	1:100	Goat serum	Rat	Anti-rat IgG
Cleaved caspase-3	9661	CST	1:100	Horse serum	Rabbit	Anti-rabbit IgG
F4/80	ab111101	Abcam	1:100	Horse serum	Rabbit	Anti-rabbit IgG
FoxP3	ab54501*	Abcam	1:450	Horse serum	Rabbit	Anti-rabbit IgG
GFP	ab183734	Abcam	1:200	Horse serum	Rabbit	Anti-rabbit IgG
Granzyme B	ab4059	Abcam	1:150	Horse serum	Rabbit	Anti-rabbit IgG
Ki67	ab16667	Abcam	1:100	Horse serum	Rabbit	Anti-rabbit IgG
NeuN	24307	CST	1:400	Horse serum	Rabbit	Anti-rabbit IgG
PD-L1	ab205921	Abcam	1:400	Horse serum	Rabbit	Anti-rabbit IgG
pSTAT1	9167	CST	1:100	Horse serum	Rabbit	Anti-rabbit IgG
T-bet	ab91109	Abcam	1:100	Horse serum	Mouse	Anti-mouse IgG

Table 1: List of primary antibodies. Primary antibodies used in this study, together with their sources, optimized working dilutions, recommended dilution buffers, host species, and appropriate secondary antibodies for single- and dual-color immunohistochemistry staining.

Conjugated Secondary Antibody	Catalog Number	Host Species	Drop/section	Vendor
HRP anti-rat IgG	MP-7444-15	Goat	One drop (undiluted)	Vector Laboratories
HRP anti-rabbit IgG	MP-7401	Horse
HRP anti-mouse IgG	MP-7402-15	Horse
AP anti-rat IgG	MP-5404-15	Goat
AP anti-rabbit IgG	MP-5401	Horse

Table 2: List of secondary antibodies. Secondary detection reagents optimized for the single- and dual-color IHC protocols, including enzyme conjugates (HRP or AP), host species, recommended application volume, and supplier information.

Substrate	Detection Enzyme	Buffer Composition	Reagent Formulation	Incubation Time	Notes
Vector Blue	Alkaline phosphatase	100 mM Tris-HCl, pH 8.2–8.5, 0.1% Tween 20	2 drops (80 µL) Reagent 1 + 2 drops (80 µL) Reagent 2 + 2 drops (45 µL) Reagent 3	20–30 min	For dual IHC blue staining; Partially soluble in xylene; xylene-free reagents and mounting media recommended
Vector Red	Alkaline phosphatase	100 mM Tris-HCl, pH 8.2–8.5, 0.1% Tween 20	2 drops (80 µL) Reagent 1 + 2 drops (80 µL) Reagent 2 + 2 drops (80 µL) Reagent 3	20–30 min	For dual IHC red staining
DAB	Horseradish peroxidase	Manufacturer-provided substrate buffer	1 drop chromogen added to 1 mL DAB substrate	Variable (see step 2.16)	Produces brown staining; carcinogenic, handle according to safety guidelines

Table 3: List of chromogenic substrates. This table summarizes the chromogenic substrates used for single- and dual-color IHC, including compatible detection enzymes, preparation and formulation, incubation times, and critical handling recommendations for optimal chromogenic signal development.

Supplementary File 1: Preparation of reagents, buffers, and substrates. This file summarizes the preparation, storage, and critical handling considerations for all reagents, buffers, blocking solutions, and chromogenic substrates used in the single- and dual-color immunohistochemistry protocols.Please click here to download this file.

Discussion

This study describes a detailed and reproducible substrate-based chromogenic IHC workflow for single- and dual-color detection of surface and intracellular antigens in FFPE brain tumor tissues derived from an orthotopic GSC model. The primary goal of this study was methodological to establish a practical, step-by-step protocol for reliable chromogenic antigen detection in immunocompetent GBM tissues.

Chromogenic IHC remains a widely used method for evaluating cellular markers in FFPE tissues because it preserves tissue architecture while allowing spatial localization of antigens within the TME^15,16. A reproducible chromogenic staining requires careful optimization of multiple technical parameters, including antigen retrieval, blocking conditions, antibody concentration, chromogen development time, and tissue handling during staining and mounting. Small deviations in these steps can result in tissue loss, weak signal intensity, excessive background staining, or poor morphological preservation. This protocol addresses common technical challenges by providing detailed procedural guidance to improve staining consistency and reproducibility.

Using the single-color IHC workflow, representative staining demonstrated successful detection of a broad range of membrane-associated and intracellular antigens, including T-cell markers, macrophage-associated markers, immune checkpoint molecules, signaling proteins, proliferation markers, and apoptosis-associated antigens. These representative results demonstrate the applicability of the protocol for detecting diverse antigen classes in FFPE GBM tissues using a standard HRP-based chromogenic detection system.

A common technical challenge in dual chromogenic IHC is the simultaneous detection of two antigens when both primary antibodies are raised in the same host species, which can complicate antibody compatibility and signal discrimination. To address this, we established a sequential AP-based dual-color staining workflow using distinct chromogenic substrates that permit clear visual separation of antigen signals under light microscopy.

Representative co-staining of CD3/Ki67 and pSTAT1/CD68 demonstrated successful simultaneous antigen detection within the same tissue section. Additional application of this workflow for NeuN/GFP co-staining further supports the versatility of the sequential dual-color protocol beyond immune marker analysis. Dual-color IHC staining can be quantified by manual scoring or digital image analysis. Common metrics include the number or percentage of single- and dual-positive cells, as well as their distribution within the tumor. When using digital analysis, consistent thresholding and region selection are recommended to ensure reproducible results.

To obtain reliable staining results with the single-color IHC protocol, a negative control omitting the primary antibody should be included. This control should produce no chromogenic signal during substrate development (step 1.15), thereby confirming the specificity of the staining procedure. For the dual-color IHC protocol, additional negative controls are recommended by omitting primary antibody I (step 2.11), primary antibody II (step 2.17), or both primary antibodies. Because both primary antibodies originate from the same host species, these controls verify that the second anti-rabbit secondary antibody (step 2.19) does not detect residual primary antibody from the first staining cycle and help exclude carryover or nonspecific chromogenic signal.

Although the representative images shown in this protocol illustrate optimal staining results, users may occasionally encounter suboptimal outcomes, including weak or absent chromogenic signal, excessive background staining, uneven staining across the tissue section, or poor signal-to-noise ratio. Such outcomes are commonly associated with inadequate antigen retrieval, suboptimal antibody concentration, insufficient blocking, overdevelopment of the chromogenic substrate, or tissue drying during the staining procedure. These issues can generally be recognized by comparison with appropriate positive and negative controls and corrected by optimizing antigen retrieval conditions, antibody dilution, blocking steps, washing procedures, and chromogen incubation times as outlined throughout the protocol.

One limitation of this study is that the applicability of single- or dual-color IHC was demonstrated using representative tissue sections from the same tumor model rather than systematically applied across multiple tumor models or tumor progression stages. However, this limitation does not affect the primary objective of this study, which was to establish and document a reproducible methodological workflow for substrate-based single- and dual-color chromogenic IHC. Compared with immunofluorescence, chromogenic IHC is compatible with routine bright-field microscopy, better preserves tissue morphology, and is less affected by tissue autofluorescence. Although optimized here for an orthotopic immunocompetent GSC-derived GBM model, this protocol may be adaptable to other preclinical tumor models and, with marker-specific optimization, to human FFPE tumor specimens. Integration with digital pathology and image analysis platforms may further expand its utility for semiquantitative tissue-based analyses.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2 L glass bottles	Corning	CORN1395-2L	Used for preparation and storage of 10× DPBS, 1× DPBS, distilled water, and sodium citrate buffer
2 L Pyrex glass beaker	Corning	1000-2L	Used for microwave-mediated antigen retrieval in sodium citrate buffer (600–800 mL working volume)
2.5% normal horse serum or normal goat serum			These come as prepared with the secondary antibodies from Vector Laboratories. Used for species-specific blocking and antibody dilution depending on the host species of the secondary antibody (anti-rabbit or anti-rat IgG) to minimize nonspecific binding and background staining
30% hydrogen peroxide (H2O2)	Fisher Scientific	BP2633500
500 mL glass bottles	VWR International	VWRU10754-818	Used for preparation and storage of flammable reagents for single-color IHC and xylene-free reagents for dual-color IHC
Absorbent bench paper with Kimwipes			Used during staining procedures to remove excess antibody or wash solution from tissue sections before subsequent incubations, minimizing reagent carryover and cross-contamination
Anti-CD3e	Abcam	ab5690
Anti-CD4	eBioscience	14-9766-80
Anti-CD68	Abcam	ab125212
Anti-CD8a	eBioscience	14-0808-80
Anti-Cleaved caspase-3	CST	9661
Anti-F4/80	Abcam	ab111101
Anti-FoxP3	Abcam	ab54501
Anti-GFP	Abcam	ab183734
Anti-Granzyme B	Abcam	ab4059
Anti-Ki67	Abcam	ab16667
Anti-NeuN	CST	24307
Anti-PD-L1	Abcam	ab205921
Anti-pSTAT1	CST	9167
Anti-T-bet	Abcam	ab91109
AP anti-rabbit IgG	Vector Laboratories	MP-5401
AP anti-rat IgG	Vector Laboratories	MP-5404-15
BLOXALL blocking solution	Vector Laboratories	SP-6000-100	Used primarily in dual-color IHC to inhibit endogenous peroxidase and alkaline phosphatase activity
Bovine serum albumin (BSA)	Bioworld	22070007-2	Used to prepare 5% universal blocking buffer to reduce nonspecific binding
Cytoseal 60 permanent mounting medium	Electron Microscopy Sciences	18006	Xylene-compatible mounting medium used for single-color IHC
DAB substrate and chromogen	DAKO	K346811-2	Used for HRP-based single-color chromogenic detection. Prepare immediately before use by adding 1 drop of chromogen to 1 mL of DAB substrate according to the manufacturer’s instructions. Unused working solution should be discarded after preparation. DAB is a potential carcinogen and should be handled in accordance with institutional biosafety and chemical safety guidelines
Distilled water			Used for reagent preparation, washes, and buffer preparation. Ultrapure or Milli-Q water may also be used
DPBS (Dulbecco’s phosphate-buffered saline)	Corning	55-031-PC
Ethanol, 200 proof	Decon Labs	2701
Fisherbrand Superfrost Plus Microscope Slides	Fisher Scientific	12-550-15	Positively charged microscopic glass slides for tissuse sections
Glass coverslips	Corning or Milipore Sigma		Appropriate sizes should be selected based on the number of tissue sections per slide. Common sizes include: 24 mm × 24 mm (single tissue section), 24 mm × 40 mm (two tissue sections), 24 mm × 50 mm (three tissue sections)
Hematoxylin	Fisher Scientific	220-102
Histo-Clear (1 L)	Electron Microscopy Sciences	64111-01	Xylene-free clearing agent used for dual-color IHC. Prepare two 500 mL bottles labeled Histo-Clear I and Histo-Clear II
HRP anti-mouse IgG	Vector Laboratories	MP-7402-15
HRP anti-rabbit IgG	Vector Laboratories	MP-7401
HRP anti-rat IgG	Vector Laboratories	MP-7444-15
Humidified chambers	Stellar Scientific	HS-120879	For incubation of tissue sections with primary and secondary antibodies
Hydrophobic barrier pen (PAP pen)	Vector Laboratories	H-4000	Used to create a water-repellent barrier around tissue sections to retain antibodies and reagents during incubation and prevent cross-contamination between multiple tissue sections on the same slide
Kimwipes	Kimtech Science	34155	Used for gentle removal of residual reagents and wash buffer from tissue sections
Magnetic stirrer	IKA	I-10001529	For preparation of sodium citrate and DPBS stock solutions
Microwave oven	Panasonic (Model: NN-L931BF)	SN: 6H61050036	2.2 cubic-foot capacity. Required for heat-mediated antigen retrieval using a 2 L glass beaker
Nikon Light Microscope	Nikon	SN: 707798	Nikon Eclipse Ci-L sn: 707798, Sola light engine 5M5-LCR-VA sn: 11787, Software: NIS Elements BR 4.60.00 & NIS Elements BR Analysis 4.60.00, Cameras: Nikon DS-Fi3 sn: 110865 (color) & Photometrics CoolSNAP DYNO snA17A608015 (B/W)
pH meter	VWR International	77619-152	For adjusting antigen retrieval buffer pH
Plastic wrap	Fisher Scientific	01810	Used to loosely cover antigen retrieval buffer during microwave heating to minimize evaporation and prevent tissue drying
Polyoxymethylene (POM) slide racks	Mopec	SP234	Compatible with the staining containers above; each rack holds up to 24 microscope slides
Precision balance	Mettler Toledo	30697420	For weighing sodium citrate and DPBS powder
Refrigerator	Thermo Scientific	TSG3005CA	For storage of temperature-sensitive reagents and antibodies
Slide storage boxes	Fisher Scientific	22-267294
Staining containers/dishes	Mopec	SP233	Each container accommodates one 24-slide staining rack
Stir bars	Fisher Scientific	14-512-128	For preparation of sodium citrate and DPBS stock solutions
Tri-sodium citrate dihydrate	Thermo Scientific Chemicals	AA3643936
Tween 20	Thermo Scientific	AAJ20605AP
VectaMount PT permanent mounting medium	Vector Laboratories	H-5600-60	Xylene-free permanent mounting medium used for dual-color IHC
Vector Blue alkaline phosphatase substrate kit	Vector Laboratories	SK-5300	Used for blue chromogenic signal development during dual-color AP-based IHC. Because Vector Blue is partially soluble in xylene, xylene-free clearing agents and mounting media must be used for dual-color staining workflows
Vector Red alkaline phosphatase substrate kit	Vector Laboratories	SK-5100	Used for red chromogenic signal development during dual-color AP-based IHC
Xylene (1 L)	StatLab	8400-1	Used as the deparaffinization and clearing agent for single-color IHC. Prepare two 500 mL bottles labeled Xylene I and Xylene II