Re: [Histonet] postnatal brain sections using vibratome

[TF]

hi, i dont have experience on vibratome section at 50 um (for electrophysiology we cut 300).
but I performed a lot on microtome or cryostat - frozen sections at 20-40 um.

if you want the brain to be "harder", perfuse longer and have better post-fixation.
unless your antigen is more than fragile and even retrieval does not work.

embeding could also be achieved by gelatin or egg yolk. Gelatin is softer, while egg yolk is harder and not transparent (you have to mark the direction).
The detach problem could be caused by the wet surface of the brain, and the agarose will fall off.


2009-03-06 



TF 



发件人： shymaa shawadfy 
发送时间： 2009-03-06  10:55:20 
收件人： histonet <@t> lists.utsouthwestern.edu 
抄送： 
主题： [Histonet] postnatal brain sections using vibratome 
 
Dear all
I am trying to use vibratome 50 祄 thick sections for immunofluorescence
using Postnatal day 0 brains. The problem is that brains are very soft and
are usually destroyed upon handling and the agarose is separated form the
brain.
My used protocol was: perfusion with 4 % PFA for 3 min, followed by several
hours to overnight post-fixation. Then embedding brains in 2 % low melting
agarose and cutting the block on vibratome using low speed.

I am thinking to add an overnight  20 % sucrose incubation step following
the post-fixation step. Then embed in agarose and continue the normal
protocol.  May be sucrose will increase the elasticity of the tissue. 

So what do you think ?


Thanks a lot
shymaa

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