[Histonet] isolated nuclei suspension for FISH

From:"Gudrun Lang"

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


We struggle around with our protocol. We take 2-3 30 Ám 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


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

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

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