Immunohistochemical Staining of CD8 Protein in Tissue Sections from Patients with Tumors

Summary

The present study presents a protocol for immunohistochemistry (IHC) that can image specific antigens located on tissue sections and determine the relative expression level of a target protein or the distribution of target cells.

Abstract

Immunohistochemistry (IHC) is one of the most useful detection techniques in scientific research and clinical practice. IHC can give researchers a direct view of a target protein or target cells through bi-colored images of histological tissue sections from patients. Cell nuclei are stained with hematoxylin and target proteins are stained via the chromogenic reaction of 3,3',4,4'-Biphenyltetramine tetrahydrochloride (DAB) in classic IHC, which can show both the relative expression level of a target protein and its location within the tissue. The principle utilized in IHC is the specific binding between an antigen and an antibody, which partially guarantees the accuracy of the results. IHC is also widely used to study cell subsets because it can show the exact location of target cell subsets in organs or tissues. This can help us understand their effects and functional mechanisms. Clinical data suggest that T-cell surface glycoprotein CD8 (CD8)+ tumor-infiltrating lymphocytes (TILs) could serve as an indication of the effectiveness of anti-programmed cell death 1 (PD-1) / programmed cell death ligand 1 (PD-L1) therapy in patients with tumors; therefore, IHC staining of CD8 protein to evaluate the CD8+ TILs in tissue sections becomes very important. IHC has several advantages. Samples are more accessible and last for a long time in storage. The reagents and equipment have been commercialized for years. However, there are also limitations. Lymphocyte infiltration into tumors is a dynamic process, and the results of IHC only reflect the infiltration at one specific time point, and not the dynamic changes over time. This disadvantage partly inhibits its clinical application in tumor immunotherapy, which mostly depends on T cell infiltration into the tumor microenvironment.

Introduction

The presence of tumor-infiltrating lymphocytes (TILs) is considered to be associated with better clinical outcomes in different kinds of cancer^1,2,3,4,5. T-cell surface glycoprotein CD8 (CD8)+ TILs are the most important effectors to prevent tumor development among all TILs^6,7,8. The application of IHC can be used to help researchers and/or pathologists accurately observe the CD8+ TILs of individual patients. Assessment of CD8+ TILs can help to determine the prognosis for patients. In addition, CD8+ TIL evaluation can be one of the indicators for tumor immunotherapy⁹.

IHC is the most commonly used detection method to evaluate specific proteins in patients’ organs or tissues, especially human tumor tissues, both qualitatively and quantitatively^10,11. IHC is widely used in pathological diagnosis. Specimen collection and storage for IHC are relatively easy. For example, there is little time limitation on the use of a sample as long as tissues are infiltrated with paraffin wax. The forms of antigen are well preserved. Thus, it is possible to handle many tissue samples at the same time. The experimental conditions can be controlled to avoid human interference. However, there are still some limitations of IHC in terms of sensitivity and background reduction.

Protocol

The Ethical Committee of the Seventh Affiliated Hospital, Sun Yat-sen University approved all experimental methods used in the study.

1. Preparation of the sample

1. Immerse the fresh human tumor tissue (within 30 min after resection; cut into 2 cm x 2 cm x 0.3 cm) in 10% formalin for 24 h (at least 2 h). The volume of formalin should be at least ten times greater than that of the tissue.
2. Set the program of the tissue processor as follows: 70% ethanol for 60 min, 80% ethanol for 60 min, 2x 95% ethanol for 60 min, 95% ethanol for 70 min, 2x 100% ethanol for 70 min, 2x xylene for 40 min, 3x paraffin wax for 40 min.
3. Immerse the paraffin-infiltrated sample with liquid wax in a mold. Cover with an embedding box. Cool the wax to immobilize the sample by putting it on an ice table (−2 °C to −8 °C). Make sure to locate the sample at the center of the mold.
4. Use a microtome to cut the wax block into 4 μm sections. Float the sections on the surface of a 55 °C water bath and mount sections on adhesive microscope slides with a positive charge. Bake the slides for at least 60 min at 65 °C to dry the slides, which can help the sections to better adhere to the slides.
5. Coat the tissue block with paraffin wax to protect the surface antigen if the samples are not to be handled immediately.

2. De-paraffinization and rehydration

1. Bake slides at 60 °C for 30 min. Wipe off the melted wax using a paper towel without touching the sections.
   NOTE: The representative slides were selected upon review of hematoxylin and eosin-stained slides, which had tumor and tumor-adjacent stroma and more TILs.
2. Immerse slides in xylene for 30 min to remove as much extra wax as possible.
3. Transfer slides through liquid based on the order below to remove all the wax in the sections and rehydrate them: 2x xylene for 10 min, 2x 100% ethanol for 5 min, 90% ethanol for 5 min, 80% ethanol for 5 min, 70% ethanol for 5 min, and deionized water for 5 min.

3. Antigen retrieval

1. In a pressure cooker, boil the tissue slides in 10 mM citrate buffer (pH 6.0) for antigen retrieval for 5 min when the cooker reaches full pressure. Do not open the pressure cooker before the temperature drops to 70 °C by water rinsing, otherwise the tissue sections may fall off.
2. Cool the slides in a water bath at room temperature for 60 min, rinse slides with phosphate-buffered saline (PBS, pH 7.5) for 5 min, and then repeat the rinsing with PBS twice.
   NOTE: The container should be placed on a shaking table when rinsing the slides.

4. Staining

1. Wipe off the liquid on the slides without touching the tissue sections. Draw a circle to surround the tissue using a hydrophobic pen to create a hydrophobic boundary.
2. Add 100 µL of 3% hydrogen peroxide onto the circle to fully cover the tissue section. Incubate the slides at room temperature for 15 min, rinse slides with PBS for 5 min, and repeat twice.
3. Add 100 µL of blocking buffer onto the circle to fully cover the tissue section. Incubate the slides at room temperature for 15 min, rinse slides with PBS for 5 min, and then repeat twice.
4. Dilute the primary CD8 protein antibody with Antibody Diluent at a ratio of 1:100. Add 100 µL of the diluted antibody onto the circle to fully cover the tissue section. Incubate the slides in a moist chamber at 4 °C overnight (about 12 h).
5. Rewarm the slides at room temperature for 30 min, rinse slides with PBS for 5 min, and then repeat twice.
6. Dilute the secondary antibody with PBS at a ratio of 1:500. Place 100 µL of the diluted antibody onto the circle to fully cover the tissue section. Incubate the slides at room temperature for 60 min, rinse slides with PBS for 5 min, and then repeat twice.
7. Dissolve the DAB particles according to the manufacturer’s instructions. Mix well before use. Add 100 µL of DAB solution onto the circle to fully cover the tissue section. The slides can be placed on white paper for better color observation, or color development under a microscope.
8. Rinse the slides with ddH₂O immediately after color development. Accurate time control can reduce background staining.
9. Immerse the slides in hematoxylin for 60 s, rinse the slides with running water for 1–2 min, and then immerse the slides in 1% hydrochloric-alcohol solution for 1 s.
10. Rinse the slides with running water for 1–2 min, immerse the slides in 0.037 M ammonium hydroxide 6x–8x, and then rinse the slides with running water for 1–2 min.
    NOTE: All incubation steps should be carried out in a moist chamber. Make sure that the liquid is well enclosed by the hydrophobic boundary, or the tissue section may dry out.

5. Dehydration and mounting sections

1. Wash slides in the following order to dehydrate the tissue sections: 80% ethanol for 5 min, 90% ethanol for 5 min, 2x 100% ethanol for 5 min, and 2x xylene for 10 min.
2. Add one drop of environmentally friendly resin to cover the section. Mount the section with a cover slip. Make sure no bubbles are trapped. Dry the slides in the air and then view them under a microscope.

Results

Successful immunohistochemical staining shows the CD8+ TILs in the tumor sections (bladder cancer). CD8 protein expression (the surface marker of CD8+ TILs) are defined and quantified using the brown signal in the image (Figure 1); the blue signal represents the cell nucleus. Meanwhile, the location of CD8+ TILs can be determined by observing the distribution of both the brown and blue signals (Figure 2). Figure 3 and Figure 4

Discussion

The presence of CD8+ TILs has been reported in different kind of tumors^6,7,8. The retrieval of the antigen denatures the CD8 protein and exposes the antigen epitope. Binding of the anti-CD8 antibody and subsequent horseradish peroxidase-labeled secondary antibody are quite effective and the color development is well established. The results of CD8 IHC can help to provide a direct view of CD8+ TILs in the tumor microenvironment, ...

Materials

Name	Company	Catalog Number	Comments
Ammonium hydroxide	Guangzhou Chemical Reagent Factory	1336-21-6
Antibody Diluent, Background Reducing	DAKO	S302281
Blocking buffer	Serotec	BUF029
CD8, Rabbit Monoclonal Antibody	Thermo Scientific	RM-9116-S
Citrate buffer	MXB Biotechnologies	MVS-0066	Antigen retrieval buffer, pH 6.0
DAB	MXB Biotechnologies	DAB-0031
Ethanol	Guangzhou Chemical Reagent Factory
Formalin	Xiuwei Commerce	XW-RS-019
Goat anti-Rabbit IgG (H+L) Cross Adsorbed Secondary Antibody, HRP conjugate	Thermo Scientific	31462
Hematoxylin	Xiuwei Commerce	XW-RS-001
Hydrochloric	Guangzhou Chemical Reagent Factory	7647-01-0
Hydrogen peroxide	Guangzhou Chemical Reagent Factory	7722-84-1
Paraffin	Leica	39601006
Phosphate-buffered saline(PBS)	MXB Biotechnologies	PBS-0060	pH 7.5
Pressure cooker	Supor
Resin	Xiuwei Commerce	XW-RS-005
Tissue processor	Leica	TP1020
Xylene	Guangzhou Chemical Reagent Factory	1330-20-7