[Histonet] IHC on free floating slices of brain

[Michelle Chang]

Dear Walter,

In order to cut the tissue on the microtome, you need to first fixed the
brain tissue (in rodents, normally, we perfused it using 4% PFA or 10%
formalin), followed by post-fixation for a couple hours depending on the
antibody of interest. Prior to cutting the tissue on the microtome, you
have to put it in 20% to 30% sucrose (to get rid of water) solution for
normally a day or two depending on how big the brain is (for mice brain
minimum 1 day and rat brain minimum 2 days).

Also note that if you have to do IHC on the free floating tissue, you will
need to incubate the primary antibody and detection system for longer time
as the tissue is thicker.

PS: You will not be able to handle free floating section that is thinner
than 25um.

Let me know if you need more information.

Michelle
michang2014 <@t> gmail.com



 Date: Wed, 20 Aug 2014 13:06:54 +0000
From: Barbara Tibbs <barbara.tibbs <@t> accuratediagnosticlabs.com>
Subject: [Histonet] RE: IHC on free floating slices of brain
To: Walter Benton <wbenton <@t> cua.md>,
        "histonet <@t> lists.utsouthwestern.edu"
        <histonet <@t> lists.utsouthwestern.edu>
Message-ID: <1408539991691.71254 <@t> accuratediagnosticlabs.com>
Content-Type: text/plain; charset="iso-8859-1"

Hello Walter,

Many years ago I did Timm's Silver stain on free-floating rat brain
slices.  The rats were perfused before removal and freezing of the brain on
dry ice.  I then would cut slices in a cryostat and float the sections in
saline until ready to stain.
I would imagine that it might be a bit costly to do IHC on free floating
tissue sections because enough reagent and antibody solutions have to be
used for each step.
Not sure I can be of any help but email if you have any questions.


Barbara S. Tibbs
Histology Supervisor
Accurate Diagnostic Labs
South Plainfield, NJ
barbara.tibbs <@t> accuratediagnosticlabs.com
732-839-3374
Cell: 610-809-6508


________________________________________
From: histonet-bounces <@t> lists.utsouthwestern.edu <
histonet-bounces <@t> lists.utsouthwestern.edu> on behalf of Walter Benton <
wbenton <@t> cua.md>
Sent: Tuesday, August 19, 2014 6:15 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] IHC on free floating slices of brain

Does anyone have information they could share on performing IHC on whole
mount, sledge microtome free floating slices of brain? A Neuropathologist
colleague is interested in doing this.

Thanks in advance for your help.

Walter Benton HT(ASCP)QIHC
Histology Supervisor
Chesapeake Urology Associates
806 Landmark Drive, Suite 127
Glen Burnie, MD 21061
443-471-5850 (Direct)
410-768-5961 (Lab)
410-768-5965 (Fax)
Chesapeakeurology.com<https://www.cua.md/owa/chesapeakeurology.com>

Voted a Best Place to Work by
Baltimore and Modern Healthcare
Magazines.