In situ Hybridization using HISTOCHOICETM MB* - Code H120, H103, H157

 

Materials

Glass slides containing tissue sections fixed in HISTOCHOICE MB

Dewaxing/rehydration series consisting of 3 staining dishes of HISTOCHOICE Clearing Agent**(H103) or xylene, 2 staining dishes of 100% ethanol, and 1 staining dish each of 95%, 70% and 50% ethanol

2X SSC, 70C

Biotin labeled probe

DAB solution (Code E885 and follow enclosed instructions)

Streptavidin-HRP conjugate

Hybridization Cocktail (45% Formamide, 1M Sodium Ion)

95, 50, 45C incubator or heating block
Dewax and rehydrate the specimens

  • Prepare the dewaxing/rehydration series (can be reused several times) while slides containing specimen sections (kept in slide boxes at -20 or -70C) are warmed to room temperature.  Also, start preheating 2X SSC to 70C.

  • Dewax slides by three incubations in dewaxer, 2 minutes each.

  • Rehydrate through the following regimen:

  • 100% ethanol-twice, 2 minutes each

  • 95% ethanol-2 minutes 

  • 70% ethanol-2 minutes

  • 50% ethanol-2 minutes

Denature the specimens:

  • Rinse slides 2 times for one minute each in pre-warmed 2X SSC buffer.

  • Heat denature slides in Hybridization Cocktail for 3 minutes at 95C.

  • Add Biotin labeled probe and continue heating at 95C for 1 min.

  • Hybridize 30-60 min. at 45C.

  • Wash 1 min. with 2X SSC at room temperature.

  • Incubate with Streptavidin-HRP for 5 min. at 50C.

  • Rinse with 1X PBS.

  • Add DAB solution and allow detection to proceed for 20 min. at 45C.

  • Rinse with 1X PBS to stop reaction.

  • Counter stain with Autohematoxylin, if desired, and mount with HISTOCHOICE Mounting Media (Code H157) or appropriate mounting media.

Notes: Digestion with protease is unnecessary when probing HISTOCHOICE fixed tissues.

            Probe concentration can be reduced by 50% in hybridizations when using  HISTOCHOICE   fixed tissues as compared to formalin fixed tissues.  

 

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Etienne Boireau - Olivier Cadas - Olivier Alméras

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