Immunohistochemistry for p53 staining in Breast Cancer Tissue
Freda Halim
Abstract
These are protocols used for study of p53 expression differences in Luminal B Her-2 negative patients. The aim of the study is to show p53 expression differences in Luminal B Her-2 negative patients with and without primary endocrine therapy resistance.We used using paraffin sections of 67 samples and stained the slides for p53 expression, using primary antibody Clone DO-7 ( DAKO).
Steps
Deparaffinization and Rehydration
Incubate slides in Xylenes for 3 minutes
Incubate slides in Xylenes for 3 minutes
Incubate slides in Xylenes for 3 minutes
Rehydrate slides in 100% Ethanol for 3 minutes
Rehydrate slides in 96% Ethanol for 3 minutes
Rehydrate slides in 70% Ethanol for 3 minutes
Rinse with running tap water and aquadest for 5 minutes
Blockage of Endogenous Peroxidase
Incubate slides in 3% H202 for 15 minutes
Rinse slides with running tap water and aquadest for 5 minutes
Antigen Retrieval
Antigen Retrieval
Antigen Retrieval with Tris EDTA ( pH9) with pressure cooker, in 950Celcius 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 ( clone DO7, Dako ) 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
