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Fish Stained With Pap: PREPARATION OF SOLUTIONS

FISH STAINED WITH PAP image

FISH STAINED WITH PAP image

FISH STAINED WITH PAP image

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.

Soak slides in Xylene for ~48 h at ambient temperature or until coverslips are easily removed or fall off of the slide.
Wash slides in fresh xylene for 5 min. I would recommend to do the third Xylene wash in fresh solution for 5 min.
Air dry slides for ~15 min
Place slides in coplin jar containing 95% Ethanol. Can be stored for several days/weeks in refrigerator.
Wash slides in coplin jar containing 85% Ethanol, followed by washing in coplin jar containing 70% ethanol, 5 min each wash.
Incubate slides in 2xSSC for 15-60 min. Proceed with pretreatment followed by FISH.

Incubate slides in 2 X SSC at 37ºC for 10 min.
Incubate slides in Pepsin Working Solution at 37ºC for 13 min.
Incubate in PBS X1 for 5 min, at ambient temperature.
Fix slides in 1% Formaldehyde for 5 min at RT.
Wash slide in PBS X 1 for 5 min at ambient temperature.
Proceed with slide denaturation according to FISH protocol.

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