[Histonet] Folding sections

[Eridana]

Hi Alexia
I am not sure exactly what you are describing but my first question would be what is your nuclear stain in? If it is an alcoholic solution the sections may get shocked and stick together.  I normally used DAPI in the secondary to counter-stain nuclei which is aqueous so that was less stress on the slices.

I  put 1-3ml of the 0.3% Triton in the mounting dish- it helped flatten and drag the slides on to the slides.

For incubations I used micro centrifuge tubes with VERY slow rotation. Primary was ON at 4o. Rinses were in dishes from Brain Research.  We changed the nets to panty hose and tightened them and glued them down with nail polish.   I usually was staining 30u sliding microtome sections not vibrotome so may not cross over.
I tried not to use paint brushes- they tended to damage the sections due to sticking to my sections.  I made bent glass rods out of pasture pipettes with a bunsen burner.

I once had the knife angle too steep on the sliding microtome, cut too fast and then had curled sections that never did flatten right.  I do not know if that is is a possibility on the vibrotome.

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, 29 Sep 2010 22:14:31 +0000 
From: Alexia Francisca N??ez Parra 	<alexia_fran <@t> hotmail.com> 
Subject: [Histonet] Free floating sections 
To: lita de foro <histonet <@t> lists.utsouthwestern.edu> 
Message-ID: <SNT129-W2203A4DC1629C55F3B778E93670 <@t> phx.gbl> 
Content-Type: text/plain; charset="iso-8859-1" 
 
 
Hello Histonetters! 
 
I am currently running a immunofluorescence reaction using free floating brain 
sections and am having problems with the sections folding as I lift them to 
mount at the end of the reaction. 
 
To obtain the sections, I first perfused the mouse with 4%PFA, dissected the 
brain and postfixed it in 4%PFA for three hours, washed it twice in PBS 
solution, and stored the brain in PBS at 4 degrees celcius. Then, I used a 
vibratome to slice the brain in 100um sections (I have also tried using a 
cryostat - and tissue tek- but this immuno runs better on sections from the 
vibratome). After being sliced, the sections were stored at 4 degrees in PBS. 
 
I am running the immuno using a culture plate with incubation with a blocker 
(10%DS in PBS-T), primary antibody incubation overnight at RT, and incubation 
with secondary antibody at RT for two hours. All these incubations are made 
under agitation. Also, I am co-staining the cells with the nuclear stain TOPRO. 
To mount the sections, I am lifting them using a thin paintbrush and placing 
them into a petri dish filled with PBS. Then I drag the sections on to a 
slide, apply Vectashield and the coverslip. Normally the sections should unfold 
as soon as they are placed in PBS 
solution, but many of them are staying folded on the brush. I have already tried 
incubating the primary antibody at 4C and I observed even more folding. 
 
Can you suggest anything to prevent this? 
 
 
 
Thank you very much 
 
Alexia Nunez-Parra 
Graduate Student 
University of Maryland