In Situ Hybridisation on formalin - fixed paraffin embedded sections

Contents:

 

Tissue Preparation (RNase Free)

  • Prepare 4% paraformaldehyde in PBS/DEPC. Place in water-bath at 65 ° C to dissolve (you need120-150 mL for 10 slides).
  • Cut 2-6um sections. Air dry sections for at least 30 minutes at room temp (best no more than 3 hours).
  • Fix sections in freshly made 4% paraformaldehyde for 10 mins.
  • Wash 3X in PBS/DEPC for 5 mins
  • Place a dish containing 300 Ml DEPC-H 2O on magnetic stirrer and slowly add 3.5 mL triethanolamine.
  • Put slides into dish (while stirring still) and add 750uL acetic anhydride drop wise over the sections, leaving stirring for 10 mins.
  • Wash 3X in PBS/DEPC for 5 mins.

 

Prehybridisation (RNase free)

  • Prepare humid chamber – wash the slide chamber with H2O and spray with RNAse Erase, cover the chamber with 3 layers of tissue papers and moisten with 5X SSC.
  • Take slides out of PBS-sol and try around the sections with tissue paper. Drew wax around the sections. Put the slides in humid chamber.
  • Add 200uL of pre-hybridisation buffer on each section.
  • Incubate in hybridisation over at 50 ° C for 2-3 hrs or at 4 ° C overnight (room temperature?).

 

Probe Preparation (RNase free)

  • Dilute the required amount of probe in 60uL of pre-hybridisation buffer
  • Heat at 85 ° C for 5 –10 mins
  • Chill in ice until needed.

 

Hybridisation (RNase free)

  • Pour off pre-hybridisation buffer from slides and dry with tissue paper
  • Add 60uL of hybridisation buffer containing probe to each section
  • Cover the section with rubber cover slip (flat side down) and using Perkin Elmer assembly tool to attach metal clip to seal section
  • Incubate at 70 ° C overnight in hybridisation oven (place paper towel under slides)

Nb. The rubber cover slips are treated with RNAse Erase, and washed in H2O/DEPC before use.

Nb. Metal clips are treated with in RNAse Erase, washed in H 2O/DEPC and dried in hybridisation oven immediately after use.

 

Posthybridisation

  • Take slides out of Hyb. Oven and remove rubber and metal clip assembly with a forceps.
  • Wash slides with 5XSSC at 70 ° C for 5 mins
  • Wash slides with 0.2XSSC at 70 ° C for 1 hr
  • Wash slides with 0.2XSSC at room temperature for 5 mins
  • Wash slides with autoclaved Maleic acid buffer at room temperature for 5 mins
  • Take slides out of Maleic acid buffer and try around the sections with tissue paper. Drew wax around the sections
  • Add 200uL of 1% blocking solution on each sections. Place the slides in humid chamber and leave at room temperature for 1 hr (Boehringer Mannheim blocking reagent is dissolved in Maleic acid buffer).

 

Antibody Binding

  • Pour off blocking solution from slides
  • Add 200uL AP-anti-DIG (at 1/2500 in blocking solution) on each section, place the slides in humid chamber and leave at room temperature for 1 hr.
  • Wash slides in Maleic acid buffer at room temperature for 10 mins.
  • Wash slides in Maleic acid buffer at room temperature for 2X 30 mins.
  • Wash slides in Buffer 3 at room temperature for 5 mins.

AP Detection

  • Take slides out Buffer 3, place in humid chamber and add 200uL of NBT/BCIP developing solution (of stock solution at 1/50 in buffer 3) on each section.
  • Incubate at room temperature overnight or until colour develops
  • Wash slides in 1mM EDTA/PBS for 10 mins
  • Take slides out of EDTA and dry with tissue paper and mount using Kaisers glycerol.

 

Solutions/Buffers

DEPC/water

  • Remove DEPC from the fridge and leave in Room Temperature for 30 mins before use
  • Add 1mL DEPC to 1 L MilliQ water, shake well, leave over night and autoclave next day

***Wear gloves and prepare in fume Hood***

 

4% Paraformaldehyde

*** Prepare RNase free and in Fume Hood ***

  • Weigh out Paraformaldehyde in a 50 mL tube.
  • Transfer paraformaldehyde into an autoclaved jar.
  • Reuse the 50 mL tube to measure and add PBS-DEPC to paraformaldehyde.

To dissolve:

  • Add 6uL 0.1 M NaOH and stir until dissolved.

 

Buffers:

  • PreHybridisation Buffer

    For 10 ml

    50% formamide (deionised)

    5 ml

    5x SSC

    2.5 ml of 20X

    1x Denhardt’s solution

    200ul 50X

    100 m g/ml herring sperm DNA

    100ul of 10 mg/ml

    100 m g/ml yeast tRNA

    100ul of 10 mg/ml

    H 20/DEPC

    2.1 ml

     

    20x SSC

    For 1 liter

    3 M Sodium Chloride (NaCl)

    175.3 g

    0.3 M tri-Sodium Citrate

    88.2 g

    pH 7.0

     

     

    Maleic Acid

    For 1 liter

    100 mM Maleic Acid

    11.6 g

    150 mM Sodium Chloride (NaCl)

    8.8 g

    pH 7.5

     

     

    Buffer B3

    For 1 liter

    100 mM Tris-HCl

     

    100 mM Sodium Chloride (NaCl)

    5.5 g

    5 mM Magnesium Chloride (MgCl2)

     

    pH 9.5