Free-floating immunostaining: tips and tricks?


#1

Recently I had to do free-floating immunostaining of spinal cords. In our lab, it’s a laborious and time-consuming procedure. For incubations and washes, most people transfer individual sections from well to well (primary ab -> wash -> wash, etc) by hand using a small hook. This gets good results but it takes forever. The upside is excellent staining and minimal use of antibody-containing solutions.

I know one can use small net-wells. I have this for 24-wells, but it uses a lot more reagent than I’d like.

Does anyone have any other recommendations? Perhaps better fitting netwells or some other kind of chambers?


#2

What is the embedding material? We use gelatin but instead of transferring to a different well we replace the solution. The net-wells can shred your sections and recovery can get frustrating. If you use a thin, soft brush to control your sections you might be able to control your sections easily without damaging them and without the risk of shredding them while pipetting out the used solution to replace it for the next wash/incubation.


#3

Thanks @lfqueme. I like the netwells but they do damage the slices.


#4

I use OCT for embedding. Sometimes pbs/5% Sucrose.