Re: [Histonet] rat brain autofluorescence

From:"Gayle Callis"



It sounds as though your fixation is very poor  per your sticky, melty 
comment.

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
HTL/HT/MT(ASCP)
Bozeman MT 59715


More results from www.clontech.com 
----- Original Message ----- 
From: "Spitzer, Nadja" 
To: 
Sent: Tuesday, April 29, 2008 7:54 AM
Subject: [Histonet] rat brain autofluorescence


Hello All,

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


~~~~~~~~~~~~~~~~~~~~~~~~
Nadja Spitzer, Ph.D.
Cell Differentiation and Development Center
Department of Biological Sciences
College of Science
Marshall University
1 John Marshall Drive
Huntington, WV, 25755
email: spitzern@marshall.edu
~~~~~~~~~~~~~~~~~~~~~~~~


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