[Histonet] RE: DAPI on thicker sections, problem with gradient

[Helen Fedor]

Hello, Maybe this method was meant to be used on Floating sections instead of mounted sections. That way the binding and penetration would go from both sides of the tissue.

Helen L. Fedor 

http://tmalab.jhmi.edu/



-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of gayle callis
Sent: Thursday, October 06, 2011 12:00 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] RE: DAPI on thicker sections, problem with gradient

Carmen, 

 

You wrote: 

 

Good morning:

 

I am Carmen Garcia from the Faculty of Medicine of the University of 

Valencia, Spain. I usually use your vectashield mounting medium for 

fluorescence with DAPI (H-1200) and I never have any problem.

But now I am working with 50um frozen sections and the problem that I 

have is that the DAPI staining does not arrive to the bottom of the 

section. Do you have any sugesstion of what can I do? (I already have 

the sections permeabilized with triton x-100) I have thougth to 

incubate with the mounting medium some hours at 4ºC or put some 

mounting medium in the antibody diluent and incubate...

 

any suggestion would be really welcome.

Thank you so much,

Kind regards,

 

Carmen

 

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***********

The problem you are experiencing is what we refer to as a DAPI staining
gradient.  Uneven staining occurs with the DAPI is not reaching all the
cells due to viscosity of the mounting media and thickness of the tissue
section.  

 

You did not say whether your thick sections have been fixed before
sectioning and staining for a tissue component, and then the DAPI - please
explain.  I suggest you do NOT use a mounting media with DAPI, but rather
make a DAPI solution which you can then apply to the section and incubate
mounting the coverslip with Vectashield that does not contain DAPI.  Using a
DAPI solution may shorten the time you now use, e.g. "some hours".    You
can buy the DAPI, ready to use from Biogenex or other vendors, but making it
in house is simple. I would assume you could add Triton X to the DAPI
solution  to improve permeability and match what you have already done with
that detergent.  After DAPI staining, mount the coverslip using  VectaShield
without DAPI.  DAPI recipes can be found on the internet, just Google DAPI.
I found it on a Johns Hopkins webpage, but IHC world may have it too.
There are other sources of ready to use DAPI if you look.  

 

We have used Prolong Gold antifade reagent with DAPI,  and get a staining
gradient on 5 um sections.   This is very annoying, and probably caused by
the actual technique of cover slipping itself. We are now going to use
Biogenex ready to use DAPI before the Prolong Gold antifade reagent without
DAPUI.   Staining for a thin section takes 5 mintues at RT, so you should
try several 50 um  sections (non experimental) to determine your optimal
staining time.   It may take only 15 minutes although cold, 4C temperature
may slow down the penetration of the stain through the thicker section.
This way you can achieve even staining of your nuclei without long
incubations in the more viscious mounting media containing DAPI. 

 

Good luck, and let us know if you have success...................

 

Gayle M. Callis

HTL/HT/MT(ASCP)

 

 

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