[Histonet] RE: IHC free floating sections

Fri Jul 31 11:05:36 CDT 2009

Mike, 

   I free float all of my rat brain sections (40-60um) and transfer them
with a paintbrush.  It takes a delicate touch to do this without causing
damage to the tissue.  I do know people who use pipettes to change the
solutions.  Either way works, it's a matter of personal preference.
There are plastic vessels with mesh screens that can be purchased
commercially, that allow you to transfer tissue without touching them.
The only problem with that method is it requires additional amounts of
antibody.  For example:  I dispense 500ul of antibody+diluent into the
well when using the paintbrush transfer method (this is done under
constant agitation on a shaker table).  The plastic tissue vessel method
requires 2 ml. of antibody+diluent (to cover tissue with enough fluid to
account for the size of the vessel within the well itself).  The link
below offers commercially made tissue transfer vessels (look under
circular staining nets and dishes).

http://www.brainresearchlab.com/productinform.html

Good luck,

Tina

Montina J. Van Meter, HT (ASCP)

Lab Manager

Autonomic Neuroscience

Pennington Biomedical Research Center

6400 Perkins Rd.

Baton Rouge, LA  70791

225-763-2564

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Mitchell
Jean A
Sent: Friday, July 31, 2009 8:30 AM
To: Michael Patrick; histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] RE: IHC free floating sections

Michael:  I use 1.5mm nichrome transfer loops for IHC staining of 50um

free floating sections and I move them into clean culture wells for each

solution.  I purchase mine from Ted Pella.  It does takes some practice

but the loops work quite well with minimal damage to the tissue.

Jean Mitchell, BS, HT (ASCP)

University of Wisconsin Hospital & Clinics

Neuromuscular Laboratory

600 Highland Avenue

Madison, WI  53792-5132 

-----Original Message-----

From: histonet-bounces <@t> lists.utsouthwestern.edu

[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Michael

Patrick

Sent: Friday, July 31, 2009 2:14 AM

To: histonet <@t> lists.utsouthwestern.edu

Subject: [Histonet] IHC free floating sections

I would like to stain free floating sections (fixed rat brain; 50um). Is

it necessary to transfer sections to "clean" culture wells with a paint

brush every time I wash or incubate with antibodies? I find this causes

significant damage to my tissue (especially since I do a lot of washes

etc.). Could I simply use a pipette to draw out the old solution and

then replace with the appropriate solution? What do most people do in

these situations?

Much thanks

Mike

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