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. Asses....

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.......

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