ß-gal Whole-mount Skin

Staining solution
930 µl 1X Staining solution
10 µl Staining supplement A
10 µl Staining supplement B
50 µl 20 mg/ml X-gal in DMF
  1. Samples are supplied in 4% PFA, rinse twice in PB for 5 minutes
  2. Prepare 1X staining solution with 900 µl dH2O & 100 µl 10X staining solution
  3. Prepare staining solution in PP tubes only!
  4. Add 1 ml staining solution to sample, incubate at 37 ºC in humidifier overnight
  5. Rinse the tissue with PBS , fix in 4% PFA overnight
  6. Rinse the tissue with PBS twice for 10 minutes each time
  7. Blot off PBS and dehydrate in 15 ml Sarstedt tubes on rocker table
  8. Microtome (4 µm) on pink superfrost slides
  9. Dry at 37 °C overnight
  10. Dewax slides
  11. Rinse in water and stain with Eosin Y (0.5% w/v in dH20 with 5% Acetic Acid) for one minute (dependant on staining)
  12. Rinse in hot water, dry and mount with fluoromount
Dehydration protocol
70% ethanol 2x 15’
98% ethanol 2x 15’
99.5% ethanol 2x 15’
Isopropanol 2x 15’
Paraffin (56-58°C) 3x 15’
Dewaxing protocol
Isopropanol
100% Ethanol
95% Ethanol
70% Ethanol
50% Ethanol