Immunohistochemistry Procedure
Cell Preparation (At least 108 cells were used per block)
1. Harvesting cells:
A. Trypsinization
B. 15 minute centrifugation at 2,500 RPM
C. PBS rinse
D. 15 minute centrifugation at 2,500 RPM
2. Suspend cells in 10 ml of 10% formaldehyde in PBS, overnight @ RT.
3. Centrifuge cells at 2,500 RPM for 10 minutes.
4. Resuspend cells in 10 ml of 70% ethanol.
5. Centrifuge cells at 2,500 RPM and taken into 70% ethanol.
Cell Staining
1. Ribbon Thickness: 5 um
2. Deparaffination Agent: Xylin
3. Hydration: Ethanol in PBS
4. Antigen Retrieval: 10 minute microwave retrieval in citrate buffer; 20 minute cooling
5. Blocking:
A. endogeneous peroxidase: 0.3% H2O2 in PBS for 10 minutes
B. endogeneous protein: 1% BSA for 20 minutes
6. Primary antibody, polyclonal anti-ATP7b (NB 100-360): 1:500, overnight @ 4 degrees Celcius
7. Secondary antibody, anti-rabbit (HRP): (dilute per manufacturer recommendation), 30 minutes @ RT
8. Wash 3x 15 minutes
9. Chromogen: AEC
10. Counterstain: Mayers hematoxylin
*The above information is only intended as a guide. The researcher should determine what protocol best meets their needs. Please follow safe laboratory procedures.
