A question for those who deal with isolated nuclei preparation for FISH.

 

We struggle around with our protocol. We take 2-3 30 µm slides,
deparaffinize them in an Eppendorf cup with Xylol, then ethanol abs., then
2xSSC at 75 degree.

Afterwards follows the digestion (Pepsin 4 mg/ml in 0,9% NaCl pH 1,5) for 25
min, 37 degree waterbath.

Then the hopefully harvested nuclei are washed in 3 times PBS, given in
0,075 M KCl, washed again and fixed in icecold aceticacid/Methanol.

 

Today we worked 5 hours on the preparation and at the end there was a tiny,
clear 1 ml solution. My colleage and me were "a little bit" disappointed
-hmm.

 

How should the solution look after the digestion? Are the slides totally
solved or can they still be seen? What is the parameter for the correct
incubation-time?

Is it very important to use a centrifuge without break?

Is the end-result turbid or clear?

What slides are to be used for spreading the nuclei: Superfrost Plus or
untreated glass slides? Wet or dry? 

 

I am glad about any hint!

 

Gudrun Lang

 

Histologie Akh Linz, Austria