Hi,

I am looking to carry out immunostaining of mauthner neurons in 4dpf larval zebrafish using the antibody 3A10 (DSHB).

I have already immunostained fixed whole-larvae using a previously established protocol (fix for two hours, wash, permeabilise, block, primary antibody incubation, wash, block, secondary antibody incubation) and managed to image the mauthner neurons.

However, I wasn't able to get it as clear of a level (i.e., wasn't able to view both mauthner neurons and its extensions) as i had hoped so my next plan is to dissect the zebrafish after fixation to enable better visualisation of the mauthner neurons.

I've come across different papers that suggest removing the eyes, cerebellum and optic tectum which I have tried with slightly improved results.

I was also wondering how best to mount the zebrafish for imaging on an inverted confocal microscope.

Any insight or protocol used in your lab would be appreciated :)

Thanks in advance!