May 29, 2023
Immunohistochemistry for FOXP3+ staining in Breast Cancer Tissue
- 1Faculty of Medicine, Pelita Harapan University, Tangerang
Protocol Citation: Freda Halim 2023. Immunohistochemistry for FOXP3+ staining in Breast Cancer Tissue. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2ly7m6elx9/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: 82571
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
Deparaffinization and Rehydration
Deparaffinization and Rehydration
23m
23m
Incubate slides in Xylenes for 3 minutes
3m
Incubate slides in Xylenes
3m
Incubate slides in Xylenes
3m
Rehydrate slides in 100% Ethanol
3m
Rehydrate slides in 96% Ethanol
3m
Rehydrate slides in 70% Ethanol
3m
Rinse with running water and aquadest
5m
Blockage of Endogenous Peroxidase
Blockage of Endogenous Peroxidase
20m
20m
Incubate slides in 3% H202
15m
Rinse slides with running tap water and aquadest
5m
Antigen Retrieval
Antigen Retrieval
1h
1h
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
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 ( Clone 236A/E7 Abcam Cambridge UK dilution 1:50 ) 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
25m
25m
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
5m
Apply Tatcha's bluing solution and rinse with running tap water and aquadest for 5 minutes
5m
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
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.