Immunohistochemistry for FOXP3+ staining in Breast Cancer Tissue
Freda Halim
Abstract
These are protocols used for study of FOXP3+ Cell Count in Luminal B Her-2 negative BC patients. We used using paraffin sections of 66 samples and stained the slides for FOXP3+ antibody, using primary antibody clone 236A/E7 Abcam Cambridge UK dilution 1:50
Steps
Deparaffinization and Rehydration
Incubate slides in Xylenes for 3 minutes
Incubate slides in Xylenes
Incubate slides in Xylenes
Rehydrate slides in 100% Ethanol
Rehydrate slides in 96% Ethanol
Rehydrate slides in 70% Ethanol
Rinse with running water and aquadest
Blockage of Endogenous Peroxidase
Incubate slides in 3% H202
Rinse slides with running tap water and aquadest
Antigen Retrieval
Antigen Retrieval with Tris EDTA ( pH9) with pressure cooker, in 950 Celcius temperature
Open the lid and cool down in room temperature
Rinse slides with running water and aquadest
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 ( Clone 236A/E7 Abcam Cambridge UK dilution 1:50 ) 120μL
Incubate for 60 minutes
Rinse with PBS
Secondary Antibody
Apply Excell Link as secondary antibody
Rinse with PBS
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 the slides
Dehydrate Slides in 70%, 96%, 100% for 5 minutes each
Incubate slides in Xylenes for 5 minutes
Mount the slides
