In Situ Hybridization

  • Fresh 10um 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 200uL of pre hybridization solution was added and incubated overnight at 4 oC.
    • 50% formamide
    • 5x SSC
    • 1x Denharts solution
    • 100ug/ml herring sperm DNA
    • 100ug/ml yeast tRNA
  • The excess solution was drained and the appropriate riboprobe was added in 60uL 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 200uL 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
  • 200uL 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).