Stained Slides before FISH: Preparation of Solutions

Preparation of Solutions
  • Make Pepsin Stock Solution. Keep frozen. Dissolve 0.5g of pepsin in 5 ml of distilled water. Concentration of pepsin is 100 mg/ml.
  • Make Pepsin Working Solution (0.05 mg/ml) Add 20 ul of Pepsin Stock Solution into 40 ml of 10mM HCl. Final concentration of pepsin is 0.05 mg/ml.
  • 1% Formaldehyde Fixative Solution (V=400 ml). Mix 10 ml 37% Formaldehyde with 386 ml PBSx1 and 4 ml 100XMgCl2 100xMgCl2 = 2 M MgCl2. Keep in refrigerator. Discard after each use. Discard solution after each use.
Coverslip Removal
  1. Soak slides in Xylene for ~48 h at ambient temperature or until coverslips are easily removed or fall off of the slide.
  2. Wash slides in fresh xylene for 5 min. Recommended to do the third Xylene wash in fresh solution for 5 min
  3. Air dry slides for ~15 min.
  4. Place slides in coplin jar containing 95% Ethanol. Can be stored for several days/weeks in refrigerator.
  5. Wash slides in coplin jar containing 85% Ethanol, followed by washing in coplin jar containing 70% ethanol, 5 min each wash.
  6. Incubate slides in 2xSSC for 15-60 min7. Proceed with pretreatment followed by FISH.
Pretreatment Procedure
  1. Incubate slides in 2 X SSC at 37ºC for 10 min.
  2. Incubate slides in Pepsin Working Solution at 37ºC for 13 min.
  3. Incubate in PBS X1 for 5 min, at ambient temperature.
  4. Fix slides in 1% Formaldehyde for 5 min at RT.
  5. Wash slide in PBS X 1 for 5 min at ambient temperature.
  6. Procede with slide denaturation according to FISH protocol

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