TITLE: Immunohistochemistry for p53 staining in Breast Cancer Tissue
AUTHORS: Freda Halim

[DESCRIPTION]
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]
SECTION: Deparaffinization and Rehydration
2. Incubate slides in Xylenes for 3 minutes

SECTION: Deparaffinization and Rehydration
1. Incubate slides in Xylenes for 3 minutes                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    
SECTION: Blockage of Endogenous Peroxidase
8. Incubate slides in 3% H202 for 15 minutes
SECTION: Deparaffinization and Rehydration
3. Incubate slides in Xylenes for 3 minutes
SECTION: Deparaffinization and Rehydration
4. Rehydrate slides in 100% Ethanol for 3 minutes
SECTION: Deparaffinization and Rehydration
5. Rehydrate slides in 96% Ethanol for 3 minutes
SECTION: Deparaffinization and Rehydration
6. Rehydrate slides in 70% Ethanol for 3 minutes

SECTION: Deparaffinization and Rehydration
7. Rinse with running tap water and aquadest for 5 minutes
SECTION: Blockage of Endogenous Peroxidase
9. Rinse slides with running tap water and aquadest for 5 minutes
SECTION: Antigen Retrieval
10. Antigen Retrieval 
SECTION: Antigen Retrieval
9.1. 
SECTION: Antigen Retrieval
10.1. Antigen Retrieval with Tris EDTA ( pH9) with pressure cooker, in 950 Celcius temperature
SECTION: Antigen Retrieval
10.2. Open the lid and cool down in room temperature 
SECTION: Antigen Retrieval
10.3. Rinse slides with running water and aquadest for 5 minutes
10.4. Rinse in PBS ( Phosphate Buffer Saline ) in pH 7.40-7.60
SECTION: Primary Antibody
11. Wipe excess liquid around the tissue
10.5. Excell Block
10.6. Rinse in PBS in pH 7.40-7.60
SECTION: Primary Antibody
12. apply primary antibody ( clone DO7, Dako ) 120μL
SECTION: Primary Antibody
13. Incubate for 60 minutes
SECTION: Primary Antibody
14. Rinse with PBS
SECTION: Secondary Antibody
15. Apply Excell Link as secondary antibody
SECTION: Secondary Antibody
16. Rinse with PBS
SECTION: Secondary Antibody
17. Apply Excell HRP as secondary antibody
SECTION: Signal Detection/ Histochemistry
18. Apply DAB ( Diamino-benzidine ) 80-100μL for 10 minutes , Rinse with running tap water and aquadest for 5 minutes
SECTION: Signal Detection/ Histochemistry
19. Apply Hematoxylline for 1 minutes, Rinse with running tap water and aquadest for 5 minutes
SECTION: Signal Detection/ Histochemistry
20. Apply Tatcha's bluing solution and rinse with running tap water and aquadest for 5 minutes
SECTION: Dehydration and Clearing
21. Clear excess water from the slides
SECTION: Dehydration and Clearing
22. Dehydrate Slides in 70%, 96%, 100% for 5 minutes each
SECTION: Dehydration and Clearing
23. Incubate slides in Xylenes for 5 minutes
SECTION: Dehydration and Clearing
24. Mount the slides