Immunohistochemistry of Paraffin Sections
100% and 95% Ethanol
Phosphate Buffered Saline (PBS)
2% Normal Goat Serum (NGS)
1% Bovine Serum Albumin (BSA)
Dilute above in PBS and filter sterilize: store at 4 C, may be kept at –20 C for long term storage
Sodium Citrate buffer : 10mM, pH 6.0. Dilute 2.941 g Sodium citrate dihydrate into 1 liter Milli-Q water and pH to 6.0
Incubate sections in 3 washes of xylene for 5 min each
Incubate sections in 2 washes of 100% ethanol for 10 min each
Incubate sections in 2 washes of 95% ethanol for 10 min each
Wash slides/sections twice with milli-Q water for 5 min each wash
Wash slide in PBS for 5 min
Immerse slides in Citrate buffer in a shallow glass dish. Cover with a glass plate and microwave for 10 min. (slides need to stay immersed in buffer at or just below boiling). Allow slides to cool for 20 min
Wash slides in milli-Q water 3 X, 5 min each wash
Wash slides in PBS for 5 min.
Incubate slides with 150 µL blocking buffer per slide for 30 min.at RT
Remove blocking buffer and rinse slides in PBS
Carefully wick off excess PBS using a kim wipe.
Dilute primary antibody in blocking buffer (1:100 - 1:1000)
Add 50 µL diluted primary Ab per slide and incubate for 1 hr @ RT.
Wash 4 X with PBS for 5 min each and wick off excess PBS.
Secondary Antibody: (Protect from light)
Dilute proper secondary Ab in blocking buffer (1:150 for Alexa fluor)
Add 50 µL diluted Ab / slide and incubate for 30 min. @ RT
Wash 4 X with PBS for 5 min. each and wick off excess PBS
Add a drop of mounting medium (80% glycerol in PBS with counter stain...DAPI, Horecst, or PI) and carefully place cover slip over section
Protect from light and visualize using the fluorescent microscope