(courtesy of Farr lab, UWMC)
Cold Acetone (-20ºC) in Coplin jars
PBS, BenchKote paper, plastic lids
Anti-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 PBS
Primary and secondary antibodies (this had a link that is broken)
DAB: 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
- Place specimens at -20ºC x20 min.
- Squeeze a dolop of OCT onto a mounting block and place frozen specimen on block.
- Let OCT harden at -20ºC x15 min.
- Set knife angle to 7.5º.
- Advancing block: Rotate wheel to bring knife forward with brake on.
- Bring block up with wheel at back
- Advance with rotator wheel
- Rotate block so tissue is perpendicular to knife
- Cut OCT block with razor to form a rectangle or a trapezoid narrower on top
- Set anti-roll device - back off it bounces by knob at bottom
- Incubate slides in cold (-20ºC) acetone x 20'.
- Tap acetone off each slide and incubate in PBS at r.t. x5'.
- 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.
- Rinse by dipping in 2 staing jars containing PBS. Soak in a 3rd PBS jar x5 min.
- Add 175 µL of 2º antibody at r.t. as before x5 min.
- Rinse with PBS as before.
- Add DAB solution at r.t. x5 min.
- Rinse in PBS 2x.
- Step slides through graded EtOH to dehydrate: 70%, 95%, 100% (x3). Toluene (or xylene) (x2).
- Add Permount and coverslip.