It sounds as though your fixation is very poor per your sticky, melty
If you flush the brain with cryoprotectant right after the fixative you
probably not allowing the PFA to crosslink the antigens with sufficient
time, and fixative is washed out by the cryoprotectant. We postfix brain
of hamsters/mice after perfusion by immersion in the fixative for additional
5 to 6 hours, maybe more would be better for you. There are those who can
guide you through rat brain fixation. The cryoprotection can be done at 4C
by immersing the brain overnight or longer AFTER the fixation is done, not
as a perfusion method. Timing on cryoprotection of a rat brain should be
forthcoming from those working with this species.
I suggest you access this free booklet in pdf form from Clontech/BD
Bioscience website. Just Google Living Colors. ®. User Manual (PT2040-1)
www.clontech.com/images/pt/dis_vectors/PT3598-5.pdf . There are excellent
troubleshooting guides for wtGFP, eGFP and other GFP chimeras. There is a
discussion of the autofluorescence patterns you will see with GFP in
tissues/cells and staining protocols.
Also, there are suggestions on how to reduce autofluorescence on the
IHCWorld website other than what you use plus a summary of methods written
up by a gentleman, found in Histonet Archives. If you want a review of
autofluorescence (privately attached) pertains directly to GFP but also
applicable to other fluorophores, I will be happy to send it to you.
Gayle M. Callis
Bozeman MT 59715
More results from www.clontech.com »
----- Original Message -----
From: "Spitzer, Nadja"
Sent: Tuesday, April 29, 2008 7:54 AM
Subject: [Histonet] rat brain autofluorescence
I have spent the last few months troubleshooting my rat brain IHC. I am new
to immuno in mammalian tissue and cobbled together a protocol from the
literature and extensive reading of the histonet archives. I'm still not
very happy with my results and was hoping that you might be able to weigh in
with opinions and/or suggestions.
I am looking at wtGFP (unfortunately I'm stuck with the wild type) and want
to combine it with IHC using the red and blue channels. My major problem has
been extreme autofluorescence of the sections in the green channel. I have
managed to get rid of the overall green background using a sodium
borohydride treatment before antibody application and the sparkly kind of
autofluorescence (lipofuscin-like?) with CuSO4 after antibodies. I am quite
happy with the reduction in autofluorescence, however, the treatments are
pretty hard on my free floating sections and many of them end up looking
pretty beat-up. Also, no-one else seems to need these treatments and I
wondered if I'm doing something wrong somewhere. The only other explanation
I can think of is that the wtGFP is less bright and so I can't turn down the
background as much when imaging?
Early in my troubleshooting I reduced fixation to perfusion with 400ml of
4%PFA followed by 200ml of 10%sucrose in PBS. The brains go straight into
30%sucrose with no post-fix. I'm having a lot of trouble now, though, with
slicing and section integrity so I think I may need to go back to a
post-fix, especially since the borohydride and copper seem to work for
getting the background down. A lot of my sections are very fragile, sticky
and sort of melt-y making me think that I've got insufficient fix.
I know I'm going on here so I'll just summarize my protocol and ask you to
please comment or suggest possible problems or solutions.
Perfuse rat via cardiac puncture; flush with PBS (I was doing 200ml but will
reduce this next time to 20-50ml); fix with 400ml PFA; flush with 10%sucrose
in PBS; remove brain and place in 30%sucrose refrigerated until it sinks.
Freeze brain in 30%sucrose at -80. Cryosection 30um sections at -17degrees
into cryoprotectant (Glycerol/Ethylene glycol/PBS); store at -20. For IHC
wash in PBS; treat sodium borohydride, wash PBS, preblock with goat serum;
primary overnight; wash and then secondary overnight; wash; CuSO4 treatment;
wash; mount on superfrost in water and coverslip with Prolong Gold.
Thanks very much I appreciate your input.
Nadja Spitzer, Ph.D.
Cell Differentiation and Development Center
Department of Biological Sciences
College of Science
1 John Marshall Drive
Huntington, WV, 25755
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