[Histonet] Re: DAPI stained smeared nuclei

[gayle callis]

Carol wrote: 

 

Someone sent me a question regarding DAPI staining ( I generally  use
Hoechst )...but anyway, they are having smearing of nuclear contents. I
would guess that is from rupturing of the nuclear membrane....over fixation?
Over air drying?  They are fixing 10 mins in cold acetone and 10 mins to air
dry. What do the experts out there say? This could be a learning moment for
me, as well.

 

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Interesting problem, but I have never had problems with DAPI staining after
either 4C acetone or my beloved acetone/alcohol fixation for murine CD
markers and other antigens that like solvent fixatives.  No problems occur
after air drying either.   We always air dry our frozen sections very well
before fixation and/or storage at -80C.   I don't think one can over air dry
unless they leave the sections out for days at RT, then antigenicity could
be damaged.     

 

I suspect there is something else going on with either how they store frozen
sections after sectioning rather than air drying, fixation in cold acetone
and then air drying again.   Possibly doing some kind of damaging thaw,
freeze, thaw?   Storing frozens in cryostat immediately after sectioning is
not advisable since condensation forms on FS when one takes them out of cold
environment.     How do they pick up the sections as I can smear a frozen
section quite thoroughly if I am not quick and tidy when mounting the FS on
a slide.   The tiniest movement will mess up tissue components, even nuclei
The very act of picking up a section on a slide can be referred to as flash
drying and is, in some ways a means of fixation although not very good.    

 

More details would help on HOW they handle the sections before fixation?
What tissues?  Cutting temperatures?   What is actually happening to their
immunofluorescence staining?   Is that fine but the nuclei are smeared?  

 

The only problem we ever had with DAPI staining is using mounting media with
DAPI which gave a staining gradient with dim staining of the nuclei in
certain areas of the tissue section.   We solved this by never using
mounting media with DAPI and now buy  DAPI solution from Biogenex or Pierce,
then stain with DAPI solution AFTER all the immunofluorescence staining was
complete followed by Prolong Gold Antifade reagent.  What kind of mounting
media do they use?  How do they rinse after all the IF staining?   Do they
use an in house prepared DAPI solution and if so, is it made correctly?   

 

Mark Tarango brought up an interesting solution to the problem, but what
happens after you pull the sections from freezer and go back to RT?    

 

Tossing out a lot of things here.........................

 

Take care

 

Gayle Callis

HTL/HT/MT(ASCP)

Bozeman MT