Dear colleagues,

I'm trying to apply ThermoFisher Donkey anti-Goat Alexa Fluor 350 secondary antibodies for immunohistochemistry of free-floating brain slices. The results are random, in most cases there is no any results, the tissue is empty. Sometimes I have fluorescent neurons but I don't understand the factor, which lead to the success. At the same time, our other secondary antibodies, Alexa Fluor 488 and 568, work well, and D a-Goat 488 is used by me as a control antibody in my trials to be sure that a first antibody works well.

My procedure is:

1. Washing first antibodies with PBS(pH7.4, 0.01M)/TBS(pH7.6,0.05M) three times 30sec/5min/10min.

2. Incubation of the tissue in the solution with secondary antibodies (DaG 350 1:200-500, 0.05% BSA, and optional Triton X100 0.05-0.5%). I put a well plate with tissue in a dark box and put it in the thermostat (37C) on a shaker and incubate it with gentle agitation during 120 min.

3. Washing secondary antibodies in a dark box with PBS(pH7.4, 0.01M)/TBS(pH7.6,0.05M) three times 30sec/5min/10min.

4. Mounting tissue slices on the slides and covering with a Sigma FluoShield solution without drying slices.

I avoid bright light during work with fluorescent antibodies but some natural light is there.

The riddle is truly frustrating I'll be glad to any advice!

A.