In Situ Hybridization with cDNA Probes
Dominik Haudenschild and David Johnson from John E. Pinar's Lab.
at CBRC
Slide Subbing
In our hands, the following procedure has lead to essentially complete retention of tissue sections during the stringent hybridization and post-hybridization washing for the in situhybridization with cRNA probes.
Preparation
Subbing solution:
1.5 g gelatin
0.3 g sodium azide
Place in bottom of Erlenmeyer flask 1L. Add about 500 mL of distilled water. Heat in nuker for a few min. Bring volume to 1 liter.
Polylysine solution:
140 mg polylysine (Sigma P-1524) in 700 mL DEPC-treated water.
Can be reused for up to 2 months at 4
200 mL DEPC-water.
100 mL Acetone
100 mL 0.2 N HCl (12.1 mL stock HCl in 700 mL distilled water).
Procedure
•Dip
slides in rack for 3 minutes each in:• 0.2 N HCl (12.1 mL stock HCl in 700 mL distilled water).
• DEPC -water.
• Acetone.
• Dry in RNA free oven (50-60oC) for 15 min. . Cool to touch after.
• Dip in subbing solution for 5 min.
• Dry in RNA free oven (50-60oC) overnight. Cool to touch.
• Immerge in polylysine solution for 10 min.
• Rinse in DEPC-water for 20 sec.
• 10 quick dips in polylysine.
• Dry overnight in oven (50-60oC) or at room temperature for 2 days.