Immunohistochemistry (IHC) of Thymus Frozen Sections

MaterialsCold Acetone (-20ºC) in Coplin jarsPBS, BenchKote paper, plastic lidsAnti-digoxigenin-HRP diluent: 1% BSA in PBS (store 10 mL aliquots of BSA at -20ºC)Anti-rat-digoxigenin diluent: 5% Milk powder, 10% mouse serum, 10% goat serum, 1% BSA in PBSPrimary and secondary antibodiesDAB: 10 mL DAB (3.8% w/v), 240 mL H2O, 13 mL Tris (1M pH 7.6), 60 µL 30% H2O2. Alternate DAB: 10 mg DAB (Sigma D5905, 10 mg/Tab, $156) 60 mL PBS, 140 µL 3% H2O2. Cutting Frozen Sections

1. Place specimens at -20ºC x20 min.
2. Squeeze a dolop of OCT onto a mounting block and place frozen specimen on block.
3. Let OCT harden at -20ºC x15 min.
4. Set knife angle to 7.5º.
5. Advancing block: Rotate wheel to bring knife forward with brake on.
6. Bring block up with wheel at back
7. Advance with rotator wheel
8. Rotate block so tissue is perpendicular to knife
9. Cut OCT block with razor to form a rectangle or a trapezoid narrower on top
10. Set anti-roll device - back off it bounces by knob at bottom

Staining Procedure

1. Incubate slides in cold (-20ºC) acetone x 20'.
2. Tap acetone off each slide and incubate in PBS at r.t. x5'.
3. Saturate BenchKote paper with PBS. Remove a slide from PBS and wipe edges of each slide with KimWipe to create a static barrier. Add 175 µL of 1º antibody to each slide. Cover with a lid x1hr.
4. Rinse by dipping in 2 staing jars containing PBS. Soak in a 3rd PBS jar x5 min.
5. Add 175 µL of 2º antibody at r.t. as before x5 min.
6. Rinse with PBS as before.
7. Add DAB solution at r.t. x5 min.
8. Rinse in PBS 2x.
9. Step slides through graded EtOH to dehydrate: 70%, 95%, 100% (x3). Toluene (or xylene) (x2).
10. Add Permount and coverslip.