Method Article

RNA In situ Hybridization with Sequential Protein Immunofluorescence in Tandem Assay

914 views

⸱

DOI:

10.3791/68615

⸱

October 10th, 2025

 ,  ,  ,  ,  ,  ,  ,  ,  ,  ,  , 
1Department of Hematopoietic Biology & Malignancy, The University of Texas MD Anderson Cancer Center, 2Department of Gynecologic Oncology and Reproductive Medicine, The University of Texas MD Anderson Cancer Center, 3Department of Leukemia, The University of Texas MD Anderson Cancer Center, 4Department of Melanoma Medical Oncology Research, The University of Texas MD Anderson Cancer Center, 5Department of Veterinary Medicine & Surgery, The University of Texas MD Anderson Cancer Center

* These authors contributed equally

In This Article

Summary

This protocol details RNA in situ hybridization with sequential protein immunofluorescence in tissue on an automated platform to characterize targeted cellular-level spatial multiple omics.

Abstract

The functionality of cells within a host does not depend on isolated signals. Instead, all components, from the smallest RNA molecules to the largest proteins, must operate in harmony and coordination within the human body. As such, the importance of approaches that integrate cellular-level spatial investigations across multiple omics is paramount to understanding cell-cell interactions and the progression of disease. Dissecting the proteome and transcriptome in the same spatial assay helps us understand how not only what a cell is being instructed to carry out (RNA), but also how it is executing those instructions in the context of the microenvironmental niche it finds itself in (protein). This manuscript is focused on integrating sequential immunofluorescence (SeqIF) with RNA in situ hybridization (ISH) with an on-tissue microfluidics driven system for high-throughput protein and RNA investigation in a spatial context (seqRNA-ISH+seqIF) that will allow up to 12 RNA and 24 protein targets in a single run with additional protein targets possible to be added via sequential runs. This method provides a sequential targeted multiomics platform that does not require consideration of fluorophore compatibility and extensive optimization for higher plexing. This allows one to understand what messages the cell is priming or is sending into its microenvironment. This targeted approach helps to validate whole transcriptome methods while examining the interactions between the RNA and proteins in a more precise manner.

Introduction

The complex functioning of cells within the human body relies on an intricate interplay of signals, from small RNA molecules to large proteins, all working in coordination1. This interaction underscores the importance of integrative approaches that examine cellular-level spatial interactions across various omics layers2,3. Such methodologies are essential for understanding the complexities of cell-cell interactions and disease progression. Many traditional methods, such as flow cytometry4 and single-cell sequencing5, cell isolates are used to investigate disaggregated samples to identify bulk ratios of phenotypes present within a specific sample. However, isolating cells for analysis may induce activation or phenotypic changes that may not accurately reflect their native biology and function in situ6. Advanced methods in spatial biology offer new strategies to study cells and their microenvironment without compromising the context of tissue architecture and spatial relationships with neighboring cells across multiple molecular types2,7.

Integrating transcriptome and proteome analyses within the same spatial assay, without isolating individual cells, offers a comprehensive view of cellular function8. This method builds on single-molecule RNA fluorescent in situ hybridization (FISH) methods, but does not include proteinase digestions which complicate sequential protein interrogations8. The flexibility of the approach to be used on a single section provides spatial higher resolution and less image processing to integrate and align sections. This approach provides insights into both the instructions received by a cell (RNA) and their execution (protein) within its specific microenvironmental niche. By combining these layers of information, researchers can gain deeper insights into cellular behavior and communication.

While manual methods exist, recent advancements in spatial omics technologies have enabled more complex analyses with a greater number of proteomic markers examined using manual multiplexing9. These approaches have included advancement in both protein and RNA-ISH assays10,11,12,13,14, and automated methodologies that require system-specific conjugated antibodies15,16,17, imaging mass cytometry (IMC)18,19,20 and other automated fluorescent sequential proteomic methodologies that utilize off-the-shelf primary and secondary antiboedies2,21,22. These technologies have facilitated either proteomics or combined transcriptomics and proteomics using targeted approaches and usually visualize these both the RNA via probes and proteins via antibodies based on similar conjugates allowing simultaneous or sequential RNA and protein to be co-detected using the same methodology or instruments. By providing a comprehensive view of cellular activity, these types of approaches aim to enhance our understanding of cellular communication between cells and their microenvironment. It offers a valuable tool for deciphering the complexities of cellular interaction, potentially revealing new insights into normal physiological processes and disease mechanisms.

This protocol provides guidance for integrating RNA in situ hybridization (ISH) with sequential immunofluorescence (SeqIF) with an on-tissue microfluidics driven system. This provides a high-throughput tool for the investigation of RNA and protein within their native spatial context. This method (seqRNA-ISH+seqIF) that will allow up to 12 RNA and 24 protein targets in a single run, with additional protein targets possible to be added via sequential runs. While this protocol focuses on one specific microfluidic system, tissue handling, RNA quality assurance, and antibody selection and titration guidelines provided can be adapted to other methodologies. However, this method uses pH 9.0 antigen retrieval for efficient, protease-free epitope retrieval, improving the proteomics stability while providing some targeted transcriptomics, unlike traditional RNA-ISH at pH 6.0 with protease to increase RNA probe accessibility but fails to preserve the proteome8. The seqRNA-ISH+seqIF method is visually outlined in Figure 1, demonstrating the RNA FISH amplification followed by cleaving before utilizing a primary-secondary antibody pairing method that utilizes a gentle chemical removal of primaries and secondaries. The sequential nature of the seqIF and detection overcomes many of the limitations related to fluorophore compatibility by utilizing sequential methodologies23. This reduces the need for extensive optimization needed in simultaneous staining in high plex methodologies that require compensation for spectral overlap23.

Access restricted. Please log in or start a trial to view this content.

Protocol

This protocol uses formalin-fixed paraffin-embedded (FFPE) tissue sections collected from treatment-naive high-grade serous ovarian carcinoma patients that underwent primary cytoreductive surgery. All clinical data were obtained from the ovarian cancer repository of the Department of Gynecologic Oncology and Reproductive Medicine under protocols approved by the University of Texas MD Anderson's Institutional Review Board. Written informed consent from the patients was obtained by front desk personnel, and the studies were conducted in accordance with recognized ethical guidelines.

NOTE: All reagents, unless otherwise stated, are diluted in nuclease-free deionized water.

1. Fix and embed samples

NOTE: The choice of fixative and fixation duration strongly impacts the performance of assays, and in general spatial assays are most often done with FFPE as it preserves pathological architecture. This protocol outlines the use of FFPE tissue, as such the following protocol is suggested for FFPE tissue samples. However, RNA quality is affected by the fixation processes, as are protein epitopes, but to a lesser extent. These fixation methods, incomplete fixation, and sample degradation can increase non-specific binding in antibody-based proteomics. Thirdly, constitutive fluorescence is typically quenched during FFPE processing, but some fluorophores will moderately persist and require testing to make sure they are quenched.

  1. Section the tissue to a thickness of ≤ 5 mm to ensure uniform fixation and to prevent fat-induced diffusion barriers. Immediately fix the sections in 10% phosphate-buffered formalin (pH 7.0) at 4 °C for 24 h to optimize RNA and protein24.
  2. Process fixed tissues in a tissue processor or manually processed using the following steps
    1. Dehydrate tissue sections in molecular grade ethanol in nuclease free water from (70%, 75%, 95% 2x, 100% 3x for 1 h each).
    2. Then, immerse the tissue in xylene 2 times for 1 h each at room temperature (~23 °C).
  3. Warm up paraffin wax to 60 °C. Submerge tissue box in paraffin wax for 2 times for 1 hour each.
  4. Place inflated tissue and orient tissues in the mold, fill with fresh paraffin wax, and cool quickly to 4 °C25.
    NOTE: The best results for RNA will be obtained by completing all steps within 48 h.

2. RNA quality assessment

  1. Use three serial sections, running either all three slides in parallel or running control slides before the targeted panel to ensure the blocking is sufficient. The paired positive control probes target housekeeping genes to assess tissue quality and spatial integrity across the tissue, which can be affected by perseverance differences. Commonly used controls include UBC (a high-expressing gene), PPIB (moderate expression), and POLR2A (low expression)26. Utilize negative control probe sets, such as Dab1 (a soil bacteria RNA) on each detection channel. Optionally, include all positive and negative control probes on the same sample alongside the target probes. In this setup, place Dab1 negative controls in channels T2-T4, assign positive controls from lowest to highest expression to T1, T5, and T9, and ensure the target probes occupy the remaining channels.
    OPTIONAL: In addition, for samples with unknown RNA quality, it can be useful to determine a Distribution Value 200 (DV200 score). DV200 scores of over 30 are strongly recommended, with higher quality samples of 50 or higher preferred27. This score is an indication of the quality of RNA, and higher sample quality increases the likelihood of RNA-ISH working effectively27.

3. RNA precautions and preparations

NOTE: It is best practice to decontaminate the area of RNases and wipe it down and then spray the working area with 70% ethanol before processing in addition to using a RNase free area, always wear clean gloves, a lab coat, and a mask to minimize introduction of RNases from skin or breath. Use only RNase-free water and consumables and avoid reaching into supply bags. These precautions, as well as keeping tissue/slides cool and dry, are essential for maintaining RNA integrity during experiments.

  1. Cut the sections onto positively charged glass slides with a microtome and place the region of interest in the center of the slide, which coordinates with the imaging area (varies by system; here, it is approximately 12.5 mm x 12.5 mm).
    NOTE: Keep in mind that the ideal thickness for each section is between 4-6 µm for FFPE tissues.
  2. Bake FFPE Slides in a dry RNAse free oven, tissue facing up, at 60 °C for a minimum of 1 h and up to overnight for fatty tissues or tissue that are otherwise prone to detachment.
  3. OPTIONAL pre-treatment
    NOTE: While pre-treatment is optional, certain steps are additionally optional as outlined below.
    1. Prepare 3% hydrogen peroxide: In a 50 mL conical tube, add 5 mL of 30% hydrogen peroxide to 45 mL of 1x PBS. Store the remaining hydrogen peroxide at room temperature (~23 °C), the lid secured with parafilm, for up to three months.
      NOTE: Hydrogen peroxide is a strong oxidizer. Keep away from combustible materials, heat sources, and metals. Use in a chemical fume hood area with personal protective equipment (PPE; e.g., goggles, gloves, and lab coat). Avoid contact with skin, eyes, and inhalation. Flush skin with water for 15 min if exposed. Seek medical help if ingested or inhaled. Higher concentrations can cause severe burns and may ignite or explode with organic materials or certain metals. Always add hydrogen peroxide to water to avoid violent reactions.
    2. Prepare 3.7% formaldehyde: In a chemical fume hood, add 1 mL of 16% formaldehyde per 3.3 mL of 1x PBS. Store formaldehyde wrapped with parafilm for up to three months.
      NOTE: Formaldehyde may cause serious eye damage, respiratory irritation, and is suspected of causing cancer. Prolonged or repeated exposure may lead to skin sensitization and allergic reactions. Use only in a chemical fume hood and wear appropriate PPE (gloves, goggles/face shield, mask, lab coat, etc.). Avoid skin contact, eye contact, and inhalation. Keep away from heat sources and open flames. Dispose of paraformaldehyde and contaminated materials as hazardous waste, and only prepare the amount needed for immediate use if possible.
    3. Deparaffinize by washing the tissue slide by placing it in 50 mL conical tubes containing the following: Xylene (40 mL) for 3 washes at 5 min each in a chemical fume hood.
      ​OPTIONAL: Instead of Xylene, a non-xylene base histological clearing agent can be used, but it will potentially leave residue from paraffin and impact image quality. This involves placing the slices in 50 mL conical tubes containing the following: Non-xylene base histological clearing agent (40 mL) for two washes at 15 min each. Sections from blocks that are made with beeswax can be difficult to dewax with non-xylene. For unknown wax/paraffin mixtures, it is recommended to use Xylene with the correct safety precautions. Xylene is a hazardous and flammable substance, and prolonged exposure may cause damage to the central nervous system, liver, and kidneys. Use only in a chemical fume hood and wear PPE (e.g., gloves, masks, and lab coats.) Dispose of xylene and contaminated materials as hazardous waste. In case of exposure, seek immediate medical attention.
    4. Re-hydrate by washing tissue slide by placing in 50 mL conical tube containing the following: 100% ethanol (40 mL) for 2 washes for 3 min each; 95% ethanol (40 mL) for 2 washes at 3 min each; 70% ethanol (40 mL) for 2 washes at 3 min each; 50% ethanol (40 mL), for 2 washes at 3 min each; 30% ethanol (40 mL) for 2 washes at 3 min each; 1x PBS wash (40 mL) for 1 wash at 2 min.
    5. Peroxidase block tissue by placing in a humidity chamber at room temperature (~23 °C) and covering the tissue with approximately 100-200 µL of 3% hydrogen peroxide, for 1 wash at 10 min then wash tissue with 1x PBS wash (40 mL), for 2 washes at 2 min each.
      NOTE: Peroxidase blocking is optional and quenches residual peroxidase enzymes in tissue to reduce non-specific staining and may reduce tissue autofluorescence. This is especially important for tissues with high autofluorescence, such as bone marrow, kidney, liver, lung, intestine, and brain, where strong background signals can interfere with imaging in the TRITC and FITC emission bands28,29,30,31,32.
    6. Perform formaldehyde fixation by placing tissue in a humidity chamber at room temperature and covering the tissue with approximately 100-200 µL of 3.7% formaldehyde, for 1 wash at 10 min, then wash tissue with 1x PBS wash (40 mL), for 2 washes at 2 min each.
      NOTE: This step is optional but recommended for high-fat tissues or other tissues that have issues adhering to glass slides.

4. Antigen retrieval

NOTE: The dewaxing and antigen retrieval method recommended and outlined here differs from the method recommended for seqRNA-ISH+seqIF by the manufacturer, which utilizes a large-capacity controlled heat antigen retrieval device.

  1. Heat Induced Antigen Retrieval (HEIR) using microwave
    1. Using a temperature-monitored microwave add sufficient commercially available pH 9.0 HEIR Buffer solution (approximately 250 mL) to cover slides in the slide holder below the thermometer if applicable.
      NOTE: Antigen retrieval for RNA is typically done at pH 6.0, however the following methodologies are for pH 9.0 for higher efficiency of antibody binding.
  2. Insert the slides in buffer solution into the microwave and counterbalance any empty chambers if applicable with double distilled water. Heat slides in microwave for 15 min at 107 °C (total time is usually 30 min for HEIR buffer to reach temperature).
  3. Cool slides by placing them under a chemical fume hood at room temperature for approximately 20 min.
  4. Wash slides in 1x PBS in a 50 mL conical tube (40 mL), for 1 wash for 2 min.
  5. Store slides in a screwcap slide mailer or 50 mL conical tube filled with 1x PBS (or a commercial multistaining buffer) and proceed immediately, or store at 4 °C for up to 7 days, but ideally less than 72 h before proceeding with seqRNA-ISH+seqIF.

5. Prepare stock solutions

  1. Prepare 40x RNA imaging buffer
    1. Prepare 40x RNA Imaging Buffer by combining 400 mg of powder RNA imaging buffer and 25 mL of nuclease free deionized water
    2. Vortex the resulting solution until fully dissolved and subsequently aliquot into 1.5 mL microcentrifuge tubes (1mL per tube)
    3. Freeze and store at -20 °C for up to three months, avoiding freeze-thaw cycles.
  2. Prepare 20x saline-sodium citrate buffer (SSC)
    1. Dissolve 157.3 g of sodium chloride (NaCl) and 88.2 g of sodium citrate (Na3C6H5O7) in 800 mL of nuclease free deionized water.
    2. Measure pH and titrate to pH 7.0 dropwise with 1 M hydrochloric acid (HCl).
    3. Autoclave to sterilize.
      NOTE: Sterile 20x SSC can also be purchased from different companies. After opening, store at room temperature to 4 °C.
    4. Prepare 4x SSC buffer by combining 1 mL of 20x SSC stock with 4 mL of Nuclease Free deionized water

6. Sequential RNA-ISH + sequential immunofluorescence

NOTE: All steps outlined herein are adapted from the manufacturer protocol for the seqRNA-ISH+seqIF outlined in the Table of Materials, as such most reagents are identified as system compatible, commercial, reagents. These reagents are identified and/or provided by the manufacturer and designed to be compatible with the system. Modifications are possible and outlined in the discussion but may require additional testing and fluidics washing to prevent buildup. The software will calculate the total volume of reagents needed after the panel and protocol has been designed and inputted. However, recommended modifications are presented. Total volume will always vary based on how many cycles and targets are used per protocol. However, the antibody and RNA-ISH dilutions will not be calculated by the system unless each individual combination is entered as a unique item in the database. For this protocol, 400 uL of antibody cocktail is required per sample slide.

  1. System preparation
    1. Prior to seqRNA-ISH+seqIF, flush all microfluidics thoroughly with nuclease free water.
    2. Spray outside of the system with RNAse decontamination solution, followed by 70% ethanol.
  2. Prepare general reagents
    1. Wash Buffer: 50 mL of 20x system compatible, commercial, multistaining buffer + 950 mL of nuclease free deionized water
    2. Water: 100 mL of nuclease free deionized water.
    3. Ethanol: 100 mL of 70% Ethanol.
    4. Imaging Buffer: 16 mL of 10x system compatible, commercial, imaging solution + 140 mL of nuclease free deionized water + 4 mL of system compatible, commercial, imaging stock.
    5. Elution Buffer: 152 of system compatible, commercial, Elution Solution 1 + 8 of system compatible, commercial, Elution Solution 2
    6. Quenching Buffer: 130 mL of nuclease free deionized water+ 14 mL of system compatible, commercial, Quenching Solution 1 + 16 mL of system compatible, commercial, Quenching Solution 2.
  3. Prepare staining reagents for the RNA-ISH protocol
    ​NOTE: This step can be completed the night before and stored at 4 °C in the dark. While manufacturer recommendations state that pooled probes can be used for up to one year, it is not recommended to pool probes more than a week ahead of time due to contamination concerns. For probes, it is best to tap or gently pipette up and down to mix and gently centrifuge them to spin down before use.
    1. Load system compatible, commercial, proteinase free permeabilization solution at 500 µL per slide in a system compatible microcentrifuge tube.
    2. Pool RNA-ISH Amplifiers based on cycle (Table 1) for a total of 500 µL per slide per each of the following in a system compatible microcentrifuge tube: i) ZZ tail trees for tails T1 to T4, ii) ZZ tail trees for tails T5 to T8, iii) ZZ tail trees for tails T9 to T 12.
    3. Pool tail tree specific, amplifier matching, fluorophores for a total of 500 µL per slide per each of the following in a system-compatible microcentrifuge tube: i) Fluorophore for tree T1 to T4, ii) Fluorophore for tree T5 to T8, iii) Fluorophore for T9 to T 12.
      NOTE: This step is light sensitive
    4. Prepare 1 mL of a 1:200 dilution of commercial DAPI (stock concentration of 1 mg/mL) in commercial multistaining buffer made with nuclease free deionized water per slide
      NOTE: The procedure was performed according to the manufacturer, which includes DAPI as part of the standard reagents. If using custom formulations of DAPI that are diluted in N,N-Dimethylformamide (DMF), dilutions may vary, and titration tests will need to be conducted. However, it is recommended to use a concentration around 1:1000-1:2000 of a 1 mg/mL stock concentration. Typically, this is at a higher-than-normal concentration than manual staining protocols, as it must last through multiple rounds of imaging and stripping. DAPI fresh-made formulations in DMF tend to be brighter than commercially available DAPI formulations. Reagents with formamide, such as DAPI, are to be allocated under a chemical fume hood. Formamide can cause reproductive toxicity and organ damage and is suspected to be carcinogenic. Utilize appropriate PPE (e.g., gloves and lab coat).
    5. Prepare RNA-ISH imaging buffer immediately prior to use with 12.125 nuclease free deionized water, 1.5 mL of 10x imaging buffer stock B and 275 µL of 40x Imaging Buffer Stock A (thawed or freshly made)
    6. Prepare up to 10 pairs of primary antibodies (the primary protein antibodies are outlined in Table 2) for seqIF according to titration tested dilutions in commercial multistaining buffer for a total of 400 µL per slide in a system compatible microcentrifuge tube. Ensure that each cycle of seqIF contains only one primary antibody per host species.
    7. Prepare secondary fluorophores (Table 3) plus DAPI according to primary antibody combinations in step 6.3.7 for seqIF according to titration-tested dilutions. Dilute in multistaining buffer at a total of 400 µL per cycle in a 50 mL conical tube with a minimum of 1 mL dead volume. Example concentrations for secondary Alexa Fluor antibodies and DAPI are as follows: 1:200 dilution for Alexa Fluor 555, 1:400 dilution for Alexa Fluor 647, 1:1800 dilution for DAPI (from 1 mg/mL DAPI in DMF)
      NOTE: This step is light sensitive. The addition of DAPI to the secondaries is not according to the manufacturer's protocol, as it can aid in maintaining DAPI expression throughout multiple cycles of seqIF, which assists in aligning images post-run. Most image processing software, such as that included with this system, utilizes the DAPI signal to confirm alignment of cyclical imaging for fluorescent colocalization.
  4. System initialization
    1. Load each of the reagents from step 6.3 in the order specified by the system. Note that generally the order will be as above, but always make sure the reagents match where they are being loaded
    2. Load the antigen retrieved slide into the holder and make sure the slide imaging window is aligned in the center of the imaging space before placing the system-compatible microfluidics chip on top and locking it into place.
      NOTE: Microfluidics chips may be reused for seqIF titration tests if cleaned and stored appropriately up to one additional time, as long as gaskets remain intact. Microfluidics chips cannot be reused if running an RNA panel due to potential contamination.
    3. Prepare Cleaving Solution by mixing 1.35 mL of 4x SSC and 150 mL of commercial 10x cleaving solution.
      NOTE: The 10x Cleaving Solution quickly oxidizes and is stored in a glass ampoule that should only be broken immediately prior to use in a seqRNA-ISH+seqIF experimental run.

7. System run

  1. Run system according to manufacturer instructions, approximate run times for two slides at a 12-plex seqRNA-ISH with a 20-plex protein seqIF is 48 h for a 12.5 cm x 12.5 cm imaging window.
  2. After the seqRNA-ISH+seqIF run is complete, the slide can be removed and stored in 1x PBS or immediately utilized for additional seqIF (for up to 40 proteins).
    NOTE: It is important to clean and flush the system microfluidics using nuclease free water before and after each run to reduce the potential for contamination.

8. OPTIONAL: Image and statistical analysis

  1. Proceed with image and statistical analysis via image analysis or pathology analysis tools.

Access restricted. Please log in or start a trial to view this content.

Results

As an example of this method, we ran the following seqRNA-ISH (Table 1) + SeqIF (Table 2 and Table 3) on two slides from a human high-grade serous ovarian carcinoma.

Figure 2 illustrates the overlay of seqRNA-ISH+seqIF on a sample of ovarian tumor tissue. Structural protein markers such as collagen 1 are utilized to identify tumor regions, while phenotypic and functional markers like CD33, CD8, and Granzyme B (GZM...

Access restricted. Please log in or start a trial to view this content.

Discussion

The most critical step in preparing and handling samples is lowering RNAse contamination. This can be achieved by utilizing masks to prevent contamination from breath and by wiping work surfaces down with an RNase decontamination solution and 70% ethanol made with nuclease-free water. For FFPE slides, it is best to cut multiple, adjacent slides from FFPE blocks for seqRNA-ISH+seqIF and store them at 4 °C with desiccant. For quality control, in addition to testing the DV200 of tissue blocks, it is recommended to run ...

Access restricted. Please log in or start a trial to view this content.

Disclosures

The authors have nothing to disclose.

Acknowledgements

This research was funded in part by the Ovarian Cancer Research Alliance (OCRA 811621 and 891490), the Sie Foundation, and the Stephanie C. Stelter Endowment Fund. This research was performed in collaboration with the Flow Cytometry and Cellular Imaging Core Facility, which is supported in part by the National Institutes of Health through M. D. Anderson's Cancer Center Support Grant P30 CA016672 and Jared Burks' NCI's Research Specialist 1 R50 CA243707-01A1.

We also would like to thank Lunaphore's Emily Martersteck for technical assistance and training.

The author(s) received a set of RNAScope targeted and control probes as part of an early access program from Bio-Techne at a discounted price, and some reagents were provided free of charge for use in this study. The manufacturer had no role in study design, data collection and analysis, or preparation of the manuscript.

Access restricted. Please log in or start a trial to view this content.

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
4',6-diamidino-2-phenylindole (DAPI)Thermo Fisher Scientific/ InvitrogenEN62248 / 62248For nuclear staining (product used during this study, 62247 has been discontinued)
50mL Conical Sterile Polypropylene Centrifuge TubesThermo Fisher Scientific339652Used for aliquots & mixed secondary solutions
70% Sterile Isopropanol AlcoholTexwipeTX3270For surface decontamination
AntibodiesVariousVariousUsed for primary targets, varies based on channel used
APOE - Host: Mouse, Clone: 960318R&DMAB41443-100(note: Specific to this study) Primary protein Antibody
aSMA - Host: Mouse, Clone: 1A4CST69319SF(note: Specific to this study) Primary protein Antibody
Autocut microtomeLeica14051956472Sectioning of tissue
CD10 - Host: Rabbit, Clone: E5P7SCST65534S(note: Specific to this study) Primary protein Antibody
CD11c - Host: Rabbit, Clone: D3V1ECST93233SF(note: Specific to this study) Primary protein Antibody
CD163 - Host: Rabbit, Clone: D6U1JCST93498S(note: Specific to this study) Primary protein Antibody
CD163 - Host: Rabbit, Clone: ja51-30Invitrogenma5-32684(note: Specific to this study) Primary protein Antibody
CD20 - Host: Mouse, Clone: L26BethylA500-017ACF(note: Specific to this study) Primary protein Antibody
CD31 - Host: Mouse, Clone: 3F8E2ProteinTech66065-2-Ig(note: Specific to this study) Primary protein Antibody
CD33 - Host: Rabbit, Clone: BLR061GBethylA700-061CF(note: Specific to this study) Primary protein Antibody
CD4 - Host: Rabbit, Clone: EPR6855Abcamab181724(note: Specific to this study) Primary protein Antibody
CD45 - Host: Rabbit, Clone: BL-178-12C7BethylA700-012(note: Specific to this study) Primary protein Antibody
CD45RO - Host: Mouse, Clone: UCHL1BethylA500-020ACF(note: Specific to this study) Primary protein Antibody
CD56 - Host: Rabbit, Clone: BLR152JBethyl99746(note: Specific to this study) Primary protein Antibody
CD66b - Host: Rabbit, Clone: BLR111HInvitrogenMA5-44413(note: Specific to this study) Primary protein Antibody
CD68 - Host: Mouse, Clone: KP-1Biolegend916104(note: Specific to this study) Primary protein Antibody
CD8 - Host: Mouse, Clone: C8/144BBiolegend372902(note: Specific to this study) Primary protein Antibody
CD86 - Host: Rabbit, Clone: E2G8PCST76755SF(note: Specific to this study) Primary protein Antibody
Col 1A1 - Host: Mouse, Clone: E3E1XCST66948S(note: Specific to this study) Primary protein Antibody
DNA LoBind Tubes 2mLEppendorf22431048Used for mixed RNA Probe solutions
Drierite 21005 Indicating DesiccantCole-Palmer21005Desiccant for Desiccator
Edge-Rite Microtome BladesThermo Fisher Scientific / Invitrogen4280LHistology - Used when sectioning samples
Ethyl Alcohol 100% (200 Proof)Pharmco111000200For deparaffinization
EZ-AR2 Elegance BufferBioGeneXHK547-XAKUsed for antigen retrieval of FFPE tissue sections
FOXP3 - Host: Rabbit, Clone: PolyclonalBethylA700-034CF(note: Specific to this study) Primary protein Antibody
Goat anti-Mouse IgG (H+L) Alexa Fluor 555Thermo Fisher Scientific/ InvitrogenA-21425(note: Specific to this study) F(ab')2-Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 555
Goat anti-Mouse IgG (H+L) Alexa Fluor 647Thermo Fisher Scientific/ InvitrogenA-48289/A-21237(note: Specific to this study) F(ab')2-Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 647
Goat anti-Rabbit IgG (H+L) Alexa Fluor 555Thermo Fisher Scientific/ InvitrogenA-21430(note: Specific to this study) F(ab')2-Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 555
Goat anti-Rabbit IgG (H+L) Alexa Fluor 647Thermo Fisher Scientific/ InvitrogenA-21246(note: Specific to this study) F(ab')2-Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 647
GZMB - Host: Rabbit, Clone: PolyclonalProteinTech13588-1-AP(note: Specific to this study) Primary protein Antibody
Hs-POLR2A-T1ACD Biotechne310457-T1Human low expressing probe
Hs-PPIB-T5ACD Biotechne313907-T5Human mid expressing probe control
Hs-UBC-T9ACD Biotechne310047-T9Human high expressing control
Hydrogen Peroxide Solution 30%Sigma-AldrichHX0640-5For Tissue preparation. Helps to block endogenous peroxidase activity and to yield highly colored products. To make 3% Hydrogen Peroxide Solution
Ker8/18 - Host: Mouse, Clone: PolyclonalProteinTech66187-1-PBS(note: Specific to this study) Primary protein Antibody
LRP5 - Host: Rabbit, Clone: HPA030505SigmaHPA030505-100UL(note: Specific to this study) Primary protein Antibody
Lunaphore 20x Multistaining BufferBio-TechneBU06Used as dilutent and for washes for the COMET
Lunaphore COMET ChipBio-TechneMK03mcirofluidics chip for COMET instrument
Lunaphore COMETBio-TechneCM10-SSeq-IF autostainer
Lunaphore Elution Buffer KitBio-TechneBU07-LSolution 1 & Solution 2 for the elution steps of the COMET
Lunaphore Imaging Buffer KitBio-TechneBU09Solute & Solvent used for imaging during the COMET run
Lunaphore Quenching BuffersBio-TechneBU08-LSolution 1 & Solution 2 for the quenching steps of the COMET
MicropipetteVariousN/Afor reagent preparation
Periostin - Host: Mouse, Clone: 1A11A3ProteinTech66491-1-PBS(note: Specific to this study) Primary protein Antibody
Pierce 16% Formaldehyde (w/v), Methanol-freeThermo Fisher Scientific28908To make 3.7% PFA for tissue preparation
Probe 320102 in (T2 channel)ACD Biotechne300040Negative control DapB in T2
Probe 320102 in (T3 channel)ACD Biotechne300040Negative control DapB in T3
Probe 320102 in (T4 channel)ACD Biotechne300040Negative control DapB in T4
RNAscope HiPlex Cleaving Stock SolutionBio-TechneP/N 324399Reagent for cleaving during RNAScope assay
RNAscope HiPlex Pro for COMET 12-plex, 20-slide KitBio-Techne322075Reagents & Buffers for RNAScope
RNAscope HiPlex Probe- Hs-APOE-T2ACD Biotechne433093-T2(note: Specific to this study) Early Access RNAscope™ HiPlex CS Probe- Hs-APOE-T2
RNAscope HiPlex Probe- Hs-ARG1-T4ACD Biotechne401581-T4(note: Specific to this study) Early Access RNAscope™ HiPlex CS Probe- Hs-ARG1-T4
RNAscope HiPlex Probe- Hs-CD274-T1ACD Biotechne600863-T1(note: Specific to this study) Early Access RNAscope™ HiPlex CS Probe- Hs-CD274-T1
RNAscope HiPlex Probe- Hs-CD40-T6ACD Biotechne445973-T6(note: Specific to this study) Early Access RNAscope™ HiPlex CS Probe- Hs-CD40-T6
RNAscope HiPlex Probe- Hs-CXCL1-01-T12ACD Biotechne1256473-T12(note: Specific to this study) Early Access RNAscope™ HiPlex CS Probe- Hs-CXCL1-01-T12
RNAscope HiPlex Probe- Hs-GZMB-T7ACD Biotechne468453-T7(note: Specific to this study) Early Access RNAscope™ HiPlex CS Probe- Hs-GZMB-T7
RNAscope HiPlex Probe- Hs-IFNG-T3ACD Biotechne310503-T3(note: Specific to this study) Early Access RNAscope™ HiPlex CS Probe- Hs-IFNG-T3
RNAscope HiPlex Probe- Hs-IL23a-T8ACD Biotechne562853-T8(note: Specific to this study) Early Access RNAscope™ HiPlex CS Probe- Hs-IL23a-T8
RNAscope HiPlex Probe- Hs-IL6-T9ACD Biotechne400883-T9(note: Specific to this study) Early Access RNAscope™ HiPlex CS Probe- Hs-IL6-T9
RNAscope HiPlex Probe- Hs-TNFa-T11ACD Biotechne310423-T11(note: Specific to this study) Early Access RNAscope™ HiPlex CS Probe- Hs-TNFa-T11
RNAscope HiPlex Probe- Hs-VEGFa-T5ACD Biotechne423163-T5(note: Specific to this study) Early Access RNAscope™ HiPlex CS Probe- Hs-VEGFa-T5
RNAscope HiPlex Probe- Hs-VISTA-T10ACD Biotechne491513-T10(note: Specific to this study) Early Access RNAscope™ HiPlex CS Probe- Hs-VISTA-T10
RNAscope HiPlex12 CS Negative Control ProbeACD Biotechne324347Full 12 plex negative control panel recommended by the manufacturer. DapB in T1-T12.
RNAscope HiPlex12 CS Positive Control Probe-HsACD Biotechne324317Full 12 plex Human positive control panel recommended by the manufacturer. Hs -RTU for following housekeeping gene in channels T1 to T12: Polr2a, PPIB, UBC, HPRT1, TUBB, RPL28, RPL5, B2M, ACTB, LDHA-O1, RPLP0-X-RPLP0P2, GAPDH.
RNAse-free waterCorning46-000-CMTo clean and prep all equipment, and to use as a dilutant when necessary
RNaseZap RNase Decontamination SolutionThermo Fisher ScientificAM9782A surface decontamination solution that destroys RNases
RNeasy FFPE KitQiagen73504 & 19093For RNA Extraction & DV200 measurement
Slide MailerSimport ScientificM9504MAfor storing slides in liquid buffer.
Surgical Design General Purpose Industrial Razor BladeThermo Fisher Scientific / Invitrogen13-812-236Histology - Used when sectioning samples
TOX 1/2 - Host: Rabbit, Clone: E613QCST62886SF(note: Specific to this study) Primary protein Antibody
Xylene Histological GradeThermo Fisher ScientificUN1307For deparaffinization

References

  1. Levine, E., Hwa, T. Small RNAs establish gene expression thresholds. Curr Opin Microbiol. 11 (6), 574-579 (2008).
  2. Ferri-Borgogno, S., et al. metabolic, and subcellular mapping of the tumor immune microenvironment via 3D targeted and non-targeted multiplex multi-omics analyses. Cancers (Basel). 16 (5), 846(2024).
  3. Carstens, J. L., et al. Spatial multiplexing and omics. Nat Rev Methods Primers. 4 (1), 1-12 (2024).
  4. Cossarizza, A., et al. Guidelines for the use of flow cytometry and cell sorting in immunological studies. Eur J Immunol. 47 (10), 1584-1797 (2017).
  5. Macosko, Z. E., et al. Highly parallel genome-wide expression profiling of individual cells using nanoliter droplets. Cell. 161 (5), 1202-1214 (2015).
  6. Nichols, A. E. C., et al. Impact of isolation method on cellular activation and presence of specific tendon cell subpopulations during in vitro culture. FASEB J. 35 (7), e21733(2021).
  7. Grünwald, D., Singer, R. H., Czaplinski, K. Methods Enzymol. , Elsevier. London. (2008).
  8. Kochan, J., Wawro, M., Kasza, A. Simultaneous detection of mRNA and protein in single cells using immunofluorescence-combined single-molecule RNA FISH. Biotechniques. 59 (4), 209-221 (2015).
  9. Shih, J. D., Waks, Z., Kedersha, N., Silver, P. A. Visualization of single mRNAs reveals temporal association of proteins with microRNA-regulated mRNA. Nucleic Acids Res. 39 (17), 7740-7749 (2011).
  10. Wang, F., et al. RNAscope. J Mol Diagn. 14 (1), 22-29 (2012).
  11. Zimmerman, S. G., Peters, N. C., Altaras, A. E., Berg, C. A. Optimized RNA ISH, RNA FISH and protein-RNA double labeling (IF/FISH) in Drosophila ovaries. Nat Protoc. 8 (11), 2158-2179 (2013).
  12. Meyer, C., Garzia, A., Tuschl, T. Simultaneous detection of the subcellular localization of RNAs and proteins in cultured cells by combined multicolor RNA-FISH and IF. Methods. 118-119, 101-110 (2017).
  13. Young, A. P., Jackson, D. J., Wyeth, R. C. A technical review and guide to RNA fluorescence in situ hybridization. PeerJ. 8, e8806(2020).
  14. Pemberton, J., Kala, S., Dikshit, A., Hupple, C. Regular and young investigator award abstracts. Thorax. 72 (Suppl 3), A1654(2017).
  15. Goltsev, Y., et al. Deep profiling of mouse splenic architecture with CODEX multiplexed imaging. Cell. 174 (4), 968-981.e15 (2018).
  16. Cheng, Y., Burrack, R. K., Li, Q. Spatially resolved and highly multiplexed protein and RNA in situ detection by combining CODEX with RNAscope in situ hybridization. J Histochem Cytochem. 70 (8), 571-581 (2022).
  17. Pourmaleki, M., et al. Tumor MHC class I expression associates with intralesional IL-2 response in melanoma. Cancer Immunol Res. 10 (3), 303-313 (2022).
  18. Johnstone, R. W., Andrew, S. M., Hogarth, M. P., Pietersz, G. A., McKenzie, I. F. C. The effect of temperature on the binding kinetics and equilibrium constants of monoclonal antibodies to cell surface antigens. Mol Immunol. 27 (4), 327-333 (1990).
  19. Porichis, F., et al. High-throughput detection of miRNAs and gene-specific mRNA at the single-cell level by flow cytometry. Nat Commun. 5 (1), 5641(2014).
  20. Hu, K., Harman, A., Baharlou, H. Imaging mass cytometry for in situ immune profiling. Methods Mol Biol. 2779, 407-423 (2024).
  21. Rivest, F., et al. Fully automated sequential immunofluorescence (seqIF) for hyperplex spatial proteomics. Sci Rep. 13 (1), 1-10 (2023).
  22. Sans, M., et al. Spatial transcriptomics of intraductal papillary mucinous neoplasms of the pancreas identifies NKX6-2 as a driver of gastric differentiation and indolent biological potential. Cancer Discov. 13 (8), 1844-1861 (2023).
  23. Hoyt, C. C. Multiplex immunofluorescence and multispectral imaging: forming the basis of a clinical test platform for immuno-oncology. Front Mol Biosci. 8, 1-12 (2021).
  24. Masuda, N., Ohnishi, T., Kawamoto, S., Monden, M., Okubo, K. Analysis of chemical modification of RNA from formalin-fixed samples and optimization of molecular biology applications for such samples. Nucleic Acids Res. 27 (22), 4436-4443 (1999).
  25. Srinivasan, M., Sedmak, D., Jewell, S. Effect of fixatives and tissue processing on the content and integrity of nucleic acids. Am J Pathol. 161 (6), 1961-1971 (2002).
  26. Bingham, V., et al. RNAscope in situ hybridization confirms mRNA integrity in formalin-fixed, paraffin-embedded cancer tissue samples. Oncotarget. 8 (55), 93392-93403 (2017).
  27. Matsubara, T., et al. DV200 index for assessing RNA integrity in next-generation sequencing. Biomed Res Int. 2020 (1), 1-6 (2020).
  28. Davis, A. S., et al. Characterizing and diminishing autofluorescence in formalin-fixed paraffin-embedded human respiratory tissue. J Histochem Cytochem. 62 (6), 405-423 (2014).
  29. Jenvey, C. J., Stabel, J. R. Autofluorescence and nonspecific immunofluorescent labeling in frozen bovine intestinal tissue sections: solutions for multicolor immunofluorescence experiments. J Histochem Cytochem. 65 (9), 531-541 (2017).
  30. Walters, D. K., Jelinek, D. F. Multiplex immunofluorescence of bone marrow core biopsies: visualizing the bone marrow immune contexture. J Histochem Cytochem. 68 (2), 99-112 (2020).
  31. Zoppolato, E., et al. Optimized immunofluorescence for liver structure analysis: enhancing 3D resolution and minimizing tissue autofluorescence. Biol Methods Protoc. 10 (1), 1-12 (2025).
  32. Zhang, Y., et al. Spectral characteristics of autofluorescence in renal tissue and methods for reducing fluorescence background in confocal laser scanning microscopy. J Fluoresc. 28 (2), 561-572 (2018).
  33. Suresh, S., O'Donnell, K. A. Translational control of immune evasion in cancer. Trends Cancer. 7 (7), 580-582 (2021).
  34. Ferri-Borgogno, S., et al. Spatial transcriptomics depict ligand-receptor cross-talk heterogeneity at the tumor-stroma interface in long-term ovarian cancer survivors. Cancer Res. 83 (9), 1503-1516 (2023).
  35. Croce, A. C., Bottiroli, G. Autofluorescence spectroscopy and imaging: a tool for biomedical research and diagnosis. Eur J Histochem. 58 (4), 1-12 (2014).
  36. Croce, A. C., Bottiroli, G. New light in flavin autofluorescence. Eur J Histochem. 59 (4), 1-10 (2015).
  37. Dubois, L., Andersson, K., Asplund, A., Björkelund, H. Evaluating real-time immunohistochemistry on multiple tissue samples, multiple targets and multiple antibody labeling methods. BMC Res Notes. 6 (1), 542(2013).
  38. Chen, A. K., Cheng, Z., Behlke, M. A., Tsourkas, A. Assessing the sensitivity of commercially available fluorophores to the intracellular environment. Anal Chem. 80 (19), 7437-7444 (2008).
  39. Krenacs, L., Krenacs, T., Stelkovics, E., Raffeld, M. Methods Mol Biol. , Humana Press. New York. (2010).
  40. Weber, J., Peng, H., Rader, C. From rabbit antibody repertoires to rabbit monoclonal antibodies. Exp Mol Med. 49 (3), e305(2017).
  41. Marlin, M. C., et al. A novel process for H&E, immunofluorescence, and imaging mass cytometry on a single slide with a concise analytics pipeline. Cytometry A. 103 (12), 1010-1018 (2023).
  42. Duncan, J. S., Elliott, K. L., Kersigo, J., Gray, B., Fritzsch, B. situ hybridization methods. , Springer. New York, NY. (2015).
  43. Kersigo, J., et al. An RNAscope whole mount approach that can be combined with immunofluorescence to quantify differential distribution of mRNA. Cell Tissue Res. 374 (2), 251-262 (2018).

Access restricted. Please log in or start a trial to view this content.

Reprints and Permissions

Request permission to reuse the text or figures of this JoVE article

Request Permission

Tags

Sequential ImmunofluorescenceSpatial TranscriptomicsMultiomics PlatformCell MicroenvironmentProtein TargetsRNA HybridizationOn Tissue MicrofluidicsCell Cell Interactions

Related Articles