The thickness of my brain section is about 500um(the whole brain has been clearing for 1 week with 37℃,25V).And I did the immunostaining with the protocol provided by the authors(primary antibody:1d/washing:12h/ secondary antibody:1d/washing:12h). And follow the staining tips,eg 37℃ on a shaker.
But it was really depressed that the penetration depth of antibody was less than 100um.Even I increased the incubating time for both primary antibody and secondary antibody(3days) ,the depth was only about 100um.
Has anyone else experienced this? or any suggestion?