RE: [Histonet] (kinda) silly questions about IHC

From:"Mitchell Jean A."

I do IHC on 50 micron frozen sections using a free floating technique
that gives great results. I use flat bottom 96 well plates for staining
- it does takes a bit of practice getting the hang of transferring the
sections from well to well without damaging them though.

If you want more details on the staining protocol feel free to contact

Jean Mitchell, BS, HT (ASCP)
University of Wisconsin Hospital & Clinics
Department of Neurology
Madison, WI

On Fri Oct 29 09:52:08 EDT 2004, Anna Elisse Beaudin 

> Hello,
>    I have a question about IHC.  I am trying to do multiple
> labeling on
> sagittal brain sections that were fixed by perfusion.  I cut 
> these
> sections on a cryostat, and I am having a lot of trouble deciding
> whether to do IHC on mounted sections vs. free-floating.  When I 
> try to
> mount fixed sections directly while sectioning, I get air bubbles
> caught between the section and the slide.. the sections just 
> don't seem
> to want to lie smoothly on the slide.  Alternatively, when I 
> stain
> free-floating sections, these long sagittal sections curl up and 
> as
> such I get extremely uneven (and ugly) staining.  My actual 
> (silly)
> question is whether it is possible to collect sections into 
> solution
> (free-floating), mount them, let them dry overnight, and then do 
> IHC on
> them.  I'm torn because I need to let the section dry a little on 
> the
> slide so that they stick, but at the same time I think it might 
> be bad
> for them to dry out?  As you can see, I am very confused, and 
> would
> really appreciate anyone's advice.  Thanks in advance for your 
> help!
> Best,
> Anna Beaudin
> Division of Nutritional Sciences
> Cornell University
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