Pretreatments

RNase pretreatment

Preparation

• thaw the RNase solution
• pre-heat the humid chamber to 37°C

Procedure

• apply 30 µl of RNase-solution onto the slide and cover with cover-slip
• incubate preparations in humid chamber for 45 min at 37°C and pre-warm 70 ml PBS in water bath at 60°C
• rinse preparations for 30 min in PBS at 60°C
• rinse slides 2 times for 5 min in PBS at room temperature

Pepsin/HCl pretreatment

Preparation

• prepare fresh 70 mL solution of Pepsin/HCl
• pre-warm water bath at 37°C /li>

Procedure

• rinse slides 2x in 0.1 N HCl at room temperature
• incubate preparations for 3 h in staining jar with Pepsin/HCl solution at 37°C
• rinse slides 2x in 0.1 N HCl and 1x in BT buffer at room temperature
• dehydrate preparations by immersion in ethanol-series (5 min each step) and air-dry

Material

• glass staining jars, water-bath at 37°C and 70°C
• 200µl pipette with autoclaved tips
• humid chamber

Chemicals and solutions

• ethanol-series: 70%, 85% und 99% methylated ethanol
• 0.1 N HCl
• PBS: 0.13 NaCl, 0.007 M NaH2PO4, 0.003 M Na2HPO4, pH 7.4
• Pepsin porcine, 17 milliAnson U/mg, 2x cryst. lyophil (SERVA 31820)
• Pepsin-solution: 1 mg Pespin/mL in 0.1 N HCl
• RNase-solution: 100 µg/ml, DNase-free in Aqua dest.