So I am doing some physiology on acutely prepared brain sections (350um) and want to preform IF on these slices following treatment + response. The one tried and true method is doing an extended incubation of the whole mount section, but this requires ~10-15 days to complete the IF, so was looking for ways to embed the acute slices and then resection them thinner.

A protocol I found embedded the slices in 3% agarose and then resectioned on a a vibratome at 50um. I have tried many times and it seems impossible to get the section to be flat (on a single z-plane) in the agarose block and so gets all wonky when sectioned again (not getting a whole slice).

Has anyone tried to section a 350um (or similar size) brain slice to be thinner? Is there some method I am missing, or is there another way that might work better? Any advice would be great! Thanks :)