[Histonet] Free floating sections

[Alexia Francisca Núñez Parra]

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