[Histonet] Flat mount eye sections

Eridana eridana <@t> cox.net
Mon Aug 9 15:26:22 CDT 2010

I have done flat retina sections- which may be what you are being asked to do which is a very specialized procedure even for most research labs.
Do you have access to a sliding microtome with a freezing stage?  Otherwise I do not know any way to do this.  If you are doing flat retina (I have done birds and rats- the rats are very difficult and require a dissecting scope to even prepare them).  I did this in a lab 15 years ago but now that I am older and do not see so well, I would use a scope for any species.
For birds or rodents, perfuse the animal usually 4%PFA, remove the eye, excise  the cornea, iris and lens and being carefully not disturbing the retina remove the vitreous humor and post fixed in 4% at 4oC overnight 
Transfer to 30% sucrose in PBS at least another 24 hours
Dissect the retina from the eyecup and trimmed it with a fresh scalpel blade to flatten it out.  I cannot remember the correct term, but pigeon and chicken eyes have a comb structure inside their eyes that must be removed before flattening. It usually took about 4 cuts of varying sizes to get a flat sample.  Looks kind of like an uneven flower with cuts from the center out.  On a few eyes they had to be cut into 2 pieces to get them flat.  On a sliding microtome with freezing stage (or with dry ice on either end of the platform, build a 30% sucrose platform bigger than the eye to be sectioned
Then with the blade in place, shave down the platform to make a flat place big enough to mount the retina.  Remove the blade for safety reasons for mounting of the retina.
Use a wooden block that is super flat covered with saran wrap.  Very carefully on one side so there were no wrinkles in the wrap, place the inside of the eye against the saran wrap (with no liquid or it will slide off) and very carefully but quickly wipe the retina onto the prepared platform and let it freeze.
Replace the blade from and drop the stage or raise the blade (depending on the microtome ) and carefully section the retina.  On a pigeon we usually got at best 3 30u sections but usually did not get the retina quite flat and got 4 sections. The sections are collected in PBS and stained in micro centrifuge tubes for various antibodies then mounted on slides after staining..

Good luck

Donna Harclerode HT,HTL,QIHC (ASCP),SLS 
Histology Core Manager 
UCSD, Dept of Pathology 
9500 Gillman Drive 
BSB 2009
San Diego, CA 92093
858 534 7438

Date: Wed, 04 Aug 2010 10:57:02 -0700 
From: <sgoebel <@t> xbiotech.com> 
Subject: [Histonet] Flat Mount 
To: histonet <@t> lists.utsouthwestern.edu 

Hello all, 
   I  am being asked to do immunostaining on fla   eyes.   First,  what is a 
flat mount and does an   on  how  to  make one?  Second, what is the methodol   
stains  on  these?   Do you have to fix them, cut them,    done this and need 
some help.  Thanks guys!! 
   Sarah Goebel, B.A., HT (ASCP) 
   [DEL: XBiot 
   8201 East Riverside Dr. 
   Austin, Texas

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