AW: [Histonet] FISH together with IF

[Gudrun Lang]

Hi Wladislav,
this technique is called FICTION. I'm sure, that you have done a literature
search on this.
If you need pepsin for your FISH depends on the fixation-status of the
tissue for permeabilization and digestion of the histon-proteins. I guess
formaldehyde fixed tissue cannot do without it.
I have no personal experience with this combined method. Perhaps you have to
omit one of the pretreatment steps of FISH. I would try it.

Slide drying: I would dry at RT or just 1-2 min at 37°C. For
immunohistochemistry you have to avoid dry tissue - hmm.
Coverslipping: and sealing with rubbercement (Fixogum)is always recommended
for denaturation and hybridization over night. (but if you have got signals,
who knows)
Perhaps my thoughts were helpful
Gudrun


-----Ursprüngliche Nachricht-----
Von: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] Im Auftrag von Vlazhs
Sokols
Gesendet: Freitag, 01. Mai 2009 20:21
An: Histonet <@t> lists.utsouthwestern.edu
Betreff: [Histonet] FISH together with IF

Dear Histonetters,

In the context of Graft-versus-Host-Disease I’m trying to couple
immunfluorescence with FISH. I agree, it is a bit daring to do FISH and IF
at once. The problem is that IF is suffering from the pre-treatment during
FISH. After 2 failed stainings I was able to state that primary antibodies
of IF couldn’t bind antigens after FISH.

The question that rose concerning this over-digestion of antigens is – could
I omit the pre-treatment with pepsin, but still but still get a satisfying
FISH?

Could you, please, look through my protocol. Maybe I’m making the same
mistakes you have also encountered.

With kind regards,

Wladislav Sokalskiy,

medical student at the University of Mainz, Germany


DAY 1:

1.       Putting the section in incubator for 10 min at 70 C.

2.       Washing in xylol for 4 x 5 min and rehydrogenating for 5 min in
Ethanol.

3.       5 min in ddH2O.

4.       For antigen retrieval: washing for 20 min at ~ 100 C in
Na-Citrat-Buffer (ph 6).

5.       6 min in Washing-Buffer (don’t know exactly what it consists of,
because it was delivered within the FISH-kit)

6.       Digestion for 2,5 min with Pepsin.

7.       6 min in Washing-Buffer

8.       Dehydrogenization with Ethanol

9.       Drying for 12 min at 37 C (is it may be better to dry at the room
temperature)

10.   Hybridizing for 10 min at 75 C and keeping the section overnight at 37
C

(I haven’t coverslipped the section as I kept it overnight. Should I? Was it
a mistake on my part?)


DAY 2:

1.       Washing the section for 5 min in TBS (with 0,05 % Tween 20)

2.       Blocking the unspecific antigens with TNT (with 0,05% Tween 20) for
30 min

3.       Adding primary antibody (diluted 1:100)

4.       Keeping incubating overnight in the moisture chamber


DAY 3:

1.       Washing 2 x 15 min in TBS (with Tween)

2.       Adding secondary antibody (Alexa Fluor 488 – diluted 1:300) and
keeping it incubating for 2 h

3.       Washing it 2 x 15 min in TBS (with Tween)

4.       Adding DAPI & covering the section with glass.
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