[Histonet] BRDU free floating vibratome sections

[Sarah Jones]

Hi Kimberle,
   I've done quite a bit of vibratome sectioning and free floating
staining.  It's quite challenging!  Two ways I can think of that might
help you.  One would be to use the Shandon coverplate system.  The
sections would be floated onto slides and then the coverplate carefully
placed on to the slide.  It then fits into a holder and it gravity feeds
the reagents over the slide.  It uses a very small amount of reagent. 
The other way is to handle the free floating sections in netwells or you
can make your own using open cassettes and small petri dishes.  The most
important steps would be making sure the rinsing steps were sufficient. 
The netwell/petri dish method would use a much greater amount of
reagent.  Hope this helps.  Sarah

Sarah Jones, HT(ASCP)
Histology Lab
Dept. of Veterinary Integrative Biosciences 
College of Veterinary Medicine
Texas A&M University
College Station, TX 77843-4458
phone: 979-845-3177
fax:  979-458-3499

>>> "Kimberle M. Jacobs" <kmjacobs <@t> vcu.edu> 08/05/04 3:32 PM >>>
Hi I have searched the archives and found a lot of discussion of BRDU,
but 
no answer to the question if there is a protocol out there for 
formaldehyde-fixed, vibratome-cut, free floating sections.  Actually we 
could mount them on slides as well, I just don't want to do the whole 
paraffin embedding thing.
Also is it really necessary to have thin sections if you are going to
use 
pepsin and acid, etc. ??

We could probably cut as thin as 20 um, but would prefer to use 40 or 60
um.

We will be doing this in pregnant ferrets (E36, gestation is ~42 days)
and 
examining the brains at ages P0-P15.

Thanks very much for any help!!

Kimberle M. Jacobs, Ph.D.
Assistant Professor
Department of Anatomy and Neurobiology
Virginia Commonwealth University
P.O. Box 980709
Richmond, VA  23298-0709
(804) 827-2135
FAX: (804) 828-9477
kmjacobs <@t> vcu.edu


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