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 cell....

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 pr....

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.......

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 b.......

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