Immunohistochemistry for CD8+ staining in Breast Cancer Tissue
Freda Halim
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
Steps
Deparaffinization and rehydration
Incubate slides in Xylenes for 3 minutes
Rehydrate slides in 100% Ethanol, 96% Ethanol, 70% Ethanol each for 3 minutes
Rinse with running tap water and aquadest
Blockage of Endogenous Peroxidase
Incubate slides in 3% H202 for 15 minutes
Rinse with running tap water and aquadest
Antigen Retrieval
Antigen Retrieval with Tris EDTA ( pH9) with pressure cooker, in 95 degree Celcius temperature
Open the lid and cool down in room temperature
Rinse slides with running water and aquadest for 5 minutes
Rinse in PBS ( Phosphate Buffer Saline ) in pH 7.40-7.60
Excell Block
Rinse in PBS in pH 7.40-7.60
Primary Antibody
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
Secondary Antibody
Apply Excell Link as secondary antibody for 15 minutes, then rinse with PBS for 5 minutes
Apply Excell HRP as secondary antibody
Signal Detection/Histochemistry
Apply DAB ( Diamino-benzidine ) 80-100μL for 10 minutes , Rinse with running tap water and aquadest for 5 minutes
Apply Hematoxylline for 1 minutes, Rinse with running tap water and aquadest for 5 minutes
Apply Tatcha's bluing solution and rinse with running tap water and aquadest for 5 minutes
Dehydration and Clearing
Clear excess water from slides and Dehydrate Slides in 70%, 96%, 100% for 5 minutes each
Incubate slides in Xylenes for 5 minutes
Mount the slides
