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FISH protocol for Paraffin Embedded Tissue

Preparing the slides
  1. Bake slides overnight at 60oC

Deparaffinizing the slides
  1. Mark areas to be hybridized with a diamond tipped scribe
  2. Immerse in xylene 3 times, 5 minutes each at RT
  3. Dehydrate slides in 100% EtOH twice, 1 minute each at RT

  1. Immerse slides in 0.2N HCl for 20 minutes at RT
  2. Immerse slides in purified water for 10 minutes at RT
  3. Immerse slides in pretreatment wash buffer (2X SSC) for 3 minutes at RT
  4. Immerse slides in pretreatment solution (1M NaSCN) at 80oC for 30 minutes*
  5. Immerse the slides in purified water for 3 minutes at RT

Protease treatment
  1. Immerse slides in protease solution at 37oC for 10-40 minutes**
  2. Immerse slides in purified water for 3 minutes at RT
  3. Dehydrate slides in 100% EtOH twice, 1 minute each at RT
  4. Air dry the slides (do not air dry for more than 1 hour)

Co-denaturation and hybridization
  1. Add 10l of probe to sample area of slide
  2. Coverslip and seal edges with rubber cement
  3. Denature for 5 minutes at 73oC and hybridize for 14-18 hrs at 37oC on Hybrite

Post Hybridization wash
  1. Remove rubber cement from slides and place in post-hybridization wash buffer (2x SSC with 0.3% NP40) at RT to remove coverslips
  2. Immerse slides in post-hybridization wash buffer at 72oC for 2 minutes

Post-post hybridization wash
  1. Wash slides in 70% EtOH for 2 min
  2. Dehydrate slides in 100% EtOH for 1 min
  3. Air dry in dark with slides in an upright position

  1. Counterstain with 10 l of DAPI and coverslip
  2. Store in dark prior to enumeration

  1. Store at 4oC when not using
  2. Allow time for slides to warm to RT before enumeration
    • add protease (25mg) to protease buffer at this time
    • **digestion time varies for each new batch of protease- at this step to check digestion add DAPI or PI, coverslip and check the digestion if the tissue needs to be digested longer, soak off coverslips in 2X SSC and put back into protease buffer. If digestion is sufficient resume with the next step

Wash Buffer
2X SSC, pH 7

Sodium Thiocyanate
Sigma S-7757
1M working solution

Protease Buffer
Vysis #30-801255
500 mL

Protease I (250mg)
Vysis #30-801260
aliquot 25mg per tube, to prevent continuous freezing and thawing of protease

Post Hybridization wash buffer
2X SSC + 0.3%NP-40, pH 7
100 mL 20x SSC
847 ml of water
3 mL of NP-40
stir and adjust to pH 7
add water up to 1000 mL

Slides used for tissue microarrays
positively charged slides from Fisher
cat # 12-550-15
Copyright © 2014 Genetic Pathology Evaluation Centre All Rights Reserved; Last modified June 18, 2014