RNAse Pretreatment for Nuclear Cresyl Violet Stain
June 2011
Pre-warm RNAse buffer and glass dishes to 37º C before starting.
Pre-filter and pre-warm CV stain and glass dishes to 65º C before starting.
Rehydrate sections in running luke-warm tap water for >30 min at RT
Rinse slides in 1x SSC for 10 min at RT
Treat slides in RNAse for 2 hr at 37º C
Rinse slides twice in 1x SSC for 20 min each at 37º C
Rinse slides in running tap water at RT
Dehydrate through alcohol series and defat through xylene, then rehydrate into running tap water
Move CV stain from 65º C to RT, stain slides for 5 min in still-warm solution
Rinse excess stain in running tap water
Dehydrate with several dips in 70% EtOH
Differentiate the stain by alternating between 95% ethanol and acid alcohol until the desired outcome is reached
Finish dehydration through alcohols into xylene
Coverslip with Permount and let dry overnight.
20x SSC
175.3 g NaCl
88.2 g Sodium citrate dihydrate
pH to 7.0 with NaOH (approximately 30 l 10 N NaOH)
bring to 1 L with H2O
dilute 20-fold before use
RNAse buffer
1 ml of 25 mg/ml RNAse A (from bovine pancreas, Sigma, #R4642)
5 ml of 1 M Tris pH 7.5
5 ml of 0.5 M EDTA pH 8.0
30 ml of 5 M NaCl
to 500 ml with H2O
Acid alcohol: 95% Ethanol + 4-5 drops of glacial acetic acid
Cresyl Violet Stain
0.5 g of Cresyl violet acetate (Sigma, #C5042)
1.25 ml of glacial acetic acid
to 500 ml with H2O
Stir on heat (60C) until majority of crystals are dissolved, if possible for several days to fully dissolve. Let the
solution cool and store in dark bottle. Reheat to 60C and filter before every use. If you still get specks of solid
on sections, use a 0.2 m bottle top filter rather than a fluted paper for better filtration.