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 top on 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
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