AW: [Histonet] FISH

Gudrun Lang gu.lang <@t>
Mon May 23 15:15:37 CDT 2011

Hi Wassan,
you may depart the FISH procedure in a tissue-dependant first part and a
probe-dependant second part.
The first part comprises the pretreatment of the tissue to retrieve and
permeabilises it. The amount of pretreatment is dependant on the
fixation-duration of the tissue.
The longer the fixation - the higher the temperature of the buffer, the
longer the incubation time in the buffer, the longer the incubation time in
the protease.

Troubleshooting in this part:
Too low temperature of some slides because of inconsistent temperature in
the waterbath. Tissueslides mounted on the glass-slides in different
Too short protease-incubation in relation to the fixation time. Usually
nuclei can be digested until you see the first tiny holes. Underdigested
nuclei are dense and look grey in the triplefilter.
If the nuclei are overdigested, they get bigger holes and fuzzy borders.
Different staining results due to variing fixation-times.

The second part comprises the stringent wash. The temperature,
buffer-quality, duration are probe-depending and are usually provided in the

Troubleshooting second part:
Too high temp leads to loosing the bound probe.
Too low temp leads to high background because of unspecifc bound probes.

With your problem I would try to prolong the digestion in 10 min steps,
until a good result is found.
Hint: standardization of fixation is the crucial point. In our lab the
tumor-block is immediately fixed in a cassette as a FISH-block. So it will
be fixed very well and withstands the harsh treatment. The disadvantage is
stronger autofluorescence.
A further hint: be sure, that the gum around the coverslip is hardened
before starting the hybridizer-protocol. 10 min drying time.

I hope this helps
Gudrun Lang

-----Ursprüngliche Nachricht-----
Von: histonet-bounces <@t>
[mailto:histonet-bounces <@t>] Im Auftrag von wassan
Gesendet: Montag, 23. Mai 2011 21:24
An: h n
Betreff: [Histonet] FISH

Dear members
We just started doing FISH using HER2 FISH pharma Dx Dako kit, Code number
on malignant breast tissues.
 I was wondering what is the success ratio or percentage for say a one
run?? my
hospital is the first in our region to start FISH  
and I am the first to run it. I have no experience  with the hybridizer
but by
reading the hybridizer manual and the kit insert i discover it is user
I run the first batch (six slides only) yesterday. Continue and finish it
only three of the slides worked.
I will start a new batch next week and I appreciate any help you may offer
avoid inaccurate result.
Thank you
Shorsh General Hospital
North of Iraq
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