May 29, 2023
Immunohistochemistry for CD8+ staining in Breast Cancer Tissue
- 1Faculty of Medicine, Pelita Harapan University, Tangerang
Protocol Citation: Freda Halim 2023. Immunohistochemistry for CD8+ staining in Breast Cancer Tissue. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l69odrlqe/v1
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 28, 2023
Last Modified: May 29, 2023
Protocol Integer ID: 82572
Abstract
These are protocols used for study of CD8+ Cell Count in Luminal B Her-2 negative BC patients. We used using paraffin sections of 66 samples and stained the slides for CD8+ antibody, using primary antibody CD8 (clone C8/144B, dilution 1:100; Dako
Deparaffinization and rehydration
Deparaffinization and rehydration
23m
23m
Incubate slides in Xylenes for 3 minutes
9m
Rehydrate slides in 100% Ethanol, 96% Ethanol, 70% Ethanol each for 3 minutes
9m
Rinse with running tap water and aquadest
5m
Blockage of Endogenous Peroxidase
Blockage of Endogenous Peroxidase
20m
20m
Incubate slides in 3% H202 for 15 minutes
15m
Rinse with running tap water and aquadest
5m
Antigen Retrieval
Antigen Retrieval
1h
1h
Antigen Retrieval with Tris EDTA ( pH9) with pressure cooker, in 95 degree Celcius temperature
20m
Open the lid and cool down in room temperature
15m
Rinse slides with running water and aquadest for 5 minutes
5m
Rinse in PBS ( Phosphate Buffer Saline ) in pH 7.40-7.60
5m
Excell Block
10m
Rinse in PBS in pH 7.40-7.60
5m
Primary Antibody
Primary Antibody
1h 5m
1h 5m
Wipe excess liquid around the tissue, apply primary antibody :CD8 (clone C8/144B, dilution 1:100; Dako
Incubate for 60 minutes and then rinse with PBS for 5 minutes
1h 5m
Secondary Antibody
Secondary Antibody
40m
40m
Apply Excell Link as secondary antibody for 15 minutes, then rinse with PBS for 5 minutes
20m
Apply Excell HRP as secondary antibody
20m
Signal Detection/Histochemistry
Signal Detection/Histochemistry
27m
27m
Apply DAB ( Diamino-benzidine ) 80-100μL for 10 minutes , Rinse with running tap water and aquadest for 5 minutes
15m
Apply Hematoxylline for 1 minutes, Rinse with running tap water and aquadest for 5 minutes
6m
Apply Tatcha's bluing solution and rinse with running tap water and aquadest for 5 minutes
6m
Dehydration and Clearing
Dehydration and Clearing
20m
20m
Clear excess water from slides and Dehydrate Slides in 70%, 96%, 100% for 5 minutes each
15m
Incubate slides in Xylenes for 5 minutes
5m
Mount the slides
Protocol references
Asano Y, Kashiwagi S, Goto W, Kurata K, Noda S, Takashima T, et al. Tumour-infiltrating CD8 to FOXP3 lymphocyte ratio in predicting treatment responses to neoadjuvant chemotherapy of aggressive breast cancer. Br J Surg. 2016;103(7):845–54.