Mar 20, 2023
Immunohistochemistry for p53 staining in Breast Cancer Tissue
- Freda Halim1,2
- 1Doctoral Degree, Faculty of Medicine, Universitas Padjajaran;
- 2Surgery Department, Faculty of Medicine, Pelita Harapan Unviersity
Protocol Citation: Freda Halim 2023. Immunohistochemistry for p53 staining in Breast Cancer Tissue. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvord7dv4o/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: March 13, 2023
Last Modified: March 20, 2023
Protocol Integer ID: 78640
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).
Deparaffinization and Rehydration
Deparaffinization and Rehydration
Incubate slides in Xylenes for 3 minutes
3m
Incubate slides in Xylenes for 3 minutes
3m
Incubate slides in Xylenes for 3 minutes
3m
Rehydrate slides in 100% Ethanol for 3 minutes
3m
Rehydrate slides in 96% Ethanol for 3 minutes
3m
Rehydrate slides in 70% Ethanol for 3 minutes
3m
Rinse with running tap water and aquadest for 5 minutes
5m
Blockage of Endogenous Peroxidase
Blockage of Endogenous Peroxidase
Incubate slides in 3% H202 for 15 minutes
15m
Rinse slides with running tap water and aquadest for 5 minutes
5m
Antigen Retrieval
Antigen Retrieval
40m
40m
Antigen Retrieval
Antigen Retrieval with Tris EDTA ( pH9) with pressure cooker, in 950 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
40m
40m
Wipe excess liquid around the tissue
apply primary antibody ( clone DO7, Dako ) 120μL
Incubate for 60 minutes
1h
Rinse with PBS
5m
Secondary Antibody
Secondary Antibody
40m
40m
Apply Excell Link as secondary antibody
15m
Rinse with PBS
5m
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 the slides
Dehydrate Slides in 70%, 96%, 100% for 5 minutes each
15m
Incubate slides in Xylenes for 5 minutes
5m
Mount the slides
Protocol references
Kikuchi, S., Nishimura, R., Osako, T., et al. Definition of p53 Overexpression and its Association with the Clinicopathological Features in Luminal/HER2-negative Breast Cancer.ANTICANCER RESEARCH 33: 3891-3898 (2013)