In Situ Hybridization
- Fresh 10µm tissue sections were cut and air dried for approximately 2 hours then fixed in 4% paraformaldehyde/PBS for 10 minutes.
- The sections were then placed in 1.2% triethanolamine and acetic anhydride was added drop wise until a concentration of 0.25% was reached.
- The slides were washed in PBS and 200µl of pre hybridization solution was added and incubated overnight at 4 oC.
- 50% formamide
- 5x SSC
- 1x Denharts solution
- 100µg/ml herring sperm DNA
- 100µg/ml yeast tRNA
- The excess solution was drained and the appropriate riboprobe was added in 60µl prehybridization solution
- The slides sealed using a Perkin Elmer assembly tool and incubated at 70oC overnight.
- After hybridization the slides were rinsed in 70oC 5x SSC and then washed in 0.2x SSC, 70oC for 1 hour.
- The slides were washed for a further 5 min with RT 0.2x SSC then in Maleic acid buffer for another 5 min. at RT.
- The sections were then incubated in 1% blocking reagent (Roche) in maleic acid buffer for 1 hour at RT.
- Excess blocking reagent was allowed to drain before adding 200µl anti digoxygenin-AP Fab fragment (Boehringer Mannheim) diluted 1/2500 in blocking solution and incubated at RT for 1 hour.
- Slides were then washed in Maleic acid buffer for 10 min then a further two 30 min washes.
- The slides were then washed in “B3” buffer pH9.5 for 5 minutes
- 100mM Tris-HCL
- 100mM NaCl
- 5mM MgCl 2
- 200µl NBT/BCIP (Boehringer Mannheim) developing solution diluted 1/50 in B3 buffer was added and the slides covered and left to develop overnight at RT.
- The reaction was stopped by washing the slides in 1mM EDTA/PBS for 10 min and the slides were mounted using Kaisers glycerol gelatin (Merck).