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Immunohistochemistry of Paraffin Sections

Solutions:
  • Xylene
  • 100% and 95% Ethanol
  • Phosphate Buffered Saline (PBS)
  • Blocking buffer:
    • 2% Normal Goat Serum (NGS)
    • 1% Bovine Serum Albumin (BSA)
    • 1% Ovalbumin
    • 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

Protocol:
Deparaffinization:
  1. Incubate sections in 3 washes of xylene for 5 min each
  2. Incubate sections in 2 washes of 100% ethanol for 10 min each
  3. Incubate sections in 2 washes of 95% ethanol for 10 min each
  4. Wash slides/sections twice with milli-Q water for 5 min each wash
  5. Wash slide in PBS for 5 min

Antigen Retrieval:
  1. 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
  2. Wash slides in milli-Q water 3 X, 5 min each wash
  3. Wash slides in PBS for 5 min.

Blocking:
  1. Incubate slides with 150 µL blocking buffer per slide for 30 min.at RT
  2. Remove blocking buffer and rinse slides in PBS
  3. Carefully wick off excess PBS using a kim wipe.

Primary Antibody:
  1. Dilute primary antibody in blocking buffer (1:100 - 1:1000)
  2. Add 50 µL diluted primary Ab per slide and incubate for 1 hr @ RT.
  3. Wash 4 X with PBS for 5 min each and wick off excess PBS.

Secondary Antibody: (Protect from light)
  1. Dilute proper secondary Ab in blocking buffer (1:150 for Alexa fluor)
  2. Add 50 µL diluted Ab / slide and incubate for 30 min. @ RT
  3. Wash 4 X with PBS for 5 min. each and wick off excess PBS
  4. Add a drop of mounting medium (80% glycerol in PBS with counter stain...DAPI, Horecst, or PI) and carefully place cover slip over section
  5. Protect from light and visualize using the fluorescent microscope