Jun 01, 2026
IHC Protocol: Immunohistochemical Staining of Paraffin-Embedded Tissue Sections for GPNMB, TFE3, PD-L1, CD3, MYC, and Ki67
- Franz Zemp1,
- Hongrui Liu1,
- Douglas Mahoney1
- 1University of Calgary
Protocol Citation: Franz Zemp, Hongrui Liu, Douglas Mahoney 2026. IHC Protocol: Immunohistochemical Staining of Paraffin-Embedded Tissue Sections for GPNMB, TFE3, PD-L1, CD3, MYC, and Ki67. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1ejnpgr2/v1
Manuscript citation:
Zemp et al. 2026. GPNMB-directed CAR T-cell therapy against MiT/TFE family fusion-driven solid tumors. Nature Cancer. Accepted.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 27, 2026
Last Modified: June 01, 2026
Protocol Integer ID: 318069
Keywords: immunohistochemical staining, immunohistochemical staining of paraffin, tissue sections for gpnmb, using horseradish peroxidase, horseradish peroxidase, following primary antibody, visualization of target protein, ki67 immunohistochemical, embedded tissue section, target protein
Abstract
Immunohistochemical (IHC) detection and visualization of target proteins in formalin-fixed, paraffin-embedded (FFPE) tissue sections using horseradish peroxidase (HRP)-based detection with 3,3’-diaminobenzidine (DAB) chromogen. This protocol is optimized for the following primary antibodies: anti-human GPNMB, TFE3, PD-L1, CD3, MYC, and Ki67.
Image Attribution
Image analysis software: ImageScope v12.2.2.5015 or QuPath v0.5.0
Guidelines
Perform all incubations in a humidified chamber to prevent drying of sections.
Optimize primary antibody dilution and incubation time for each tissue type and antibody lot.
Include positive and negative controls (e.g., isotype control or omission of primary antibody) in every run.
All steps involving Dako and Agilent reagents should follow the manufacturer’s recommendations.
DAB is a hazardous chemical; handle with appropriate personal protective equipment and dispose of waste according to local regulations.
This protocol is designed for manual staining; automated stainers may require minor adjustments.
Materials
Xylene (histological grade)
Ethanol (100%, 95%, 70%, distilled water for rehydration gradient)
1× Antigen Retrieval Buffer (100X Citrate Buffer, Abcam ab93678)
Peroxidase-Blocking Solution (Dako, cat. no. S202386-2)
Protein Block (Agilent, cat. no. X090930-2)
SignalStain® Antibody Diluent (Cell Signaling, 8112)
Primary antibodies (listed above with specified dilutions)
1× TBS buffer (Santa Cruz, cat. no. sc-362305)
1× TBS-T (1× TBS + 0.01% Tween-20; Sigma)
Polymer-HRP secondary antibody (Vector Laboratories or DAKO EnVision system; species-appropriate)
DAB reagent (Dako; 1 drop DAB concentrate in 1 mL DAB substrate solution)
Hematoxylin (for counterstaining)
Cytoseal XYL mounting medium (Epredia 8312-4)
Pressure cooker (for heat-induced epitope retrieval)
Humidified incubation chamber
Slide racks and staining dishes
Aperio Scanscope® XT slide scanner (Aperio Inc.)
Safety warnings
DAB is a hazardous chemical; handle with appropriate personal protective equipment and dispose of waste according to local regulations.
Ethics statement
- Tissue/blood collection for the protocol requires prior approval by the users' Institutional Ethics Board or equivalent ethics committee
Procedure
Deparaffinization and Rehydration. Deparaffinize tissue sections in xylene (3 changes, 5 minutes each). Rehydrate through a graded ethanol series: 100% ethanol (2 changes, 3 minutes each), 95% ethanol (3 minutes), 70% ethanol (3 minutes), and finally distilled water (2 minutes).
Heat-Induced Epitope Retrieval (HIER). Place slides in 1× antigen retrieval buffer (10 mM citrate buffer, pH 6.0). Perform antigen retrieval in a pressure cooker for 22 minutes at full pressure. Allow slides to cool to room temperature in the buffer (approximately 20–30 minutes). Rinse slides in distilled water followed by 1× TBST.
Endogenous Peroxidase Blocking. Apply 1 drop of Peroxidase-Blocking Solution (Dako, S202386-2) to each section. Incubate for 15 minutes at room temperature in humidified incubation chamber. Rinse slides thoroughly with 1× TBS.
Non-Specific Binding Block. Apply 200 µL of Protein Block (Agilent, X090930-2) to each section. Incubate for 30 minutes at room temperature. Do not rinse; gently tap off excess block or blot around the section.
Primary Antibody Incubation. Dilute the appropriate primary antibody in Antibody Diluent (or 1× TBS + 1% BSA if not specified):
GPNMB (AF2550): 1:250
GPNMB (E4D7P): 1:500
TFE3 (MRQ-37): 1:100
PD-L1 (E1L3N): 1:200
CD3 (SP7): 1:100
MYC (71D10): 1:200
Ki67 (D3B5): 1:500
Apply 100–200 µL of diluted primary antibody to each section. Incubate for 1 hour at room temperature or overnight at 4°C in a humidified chamber. Wash slides 3× in 1× TBST (5 minutes each).
Secondary Antibody Incubation. Apply 1 drop of the appropriate species-specific polymer-HRP secondary antibody (Vector Laboratories or DAKO EnVision). Incubate for 30 minutes at room temperature. Wash slides 3× in 1× TBST for 5 minutes each.
Chromogenic Detection. Prepare fresh DAB working solution (1 drop DAB concentrate in 1 mL DAB substrate solution). Apply DAB solution to each section and incubate until brown color develops (monitor under microscope; typically 1–5 minutes). Stop the reaction by rinsing slides in distilled water.
Counterstaining and Dehydration. Counterstain sections with hematoxylin (1–5 minutes, depending on desired intensity). Rinse in running tap water or bluing reagent. Dehydrate through graded ethanol: 70%, 95%, 100% (2 changes, 2 minutes each). Clear in xylene (2 changes, 3 minutes each).
Mounting. Mount slides with Cytoseal XYL mounting medium and coverslips. Allow slides to dry completely.
Slide Scanning. Scan stained slides using the Aperio Scanscope® XT slide scanner at 20× or 40× magnification.
Image Analysis. Analyze digitized images using ImageScope software (v12.2.2.5015) or QuPath software (v0.5.0) for quantitative scoring of staining intensity, percentage of positive cells, and localization.