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Host species: Donkey
The lacrimal glands were taken from female Balb/C mice. Fixation is typically done over night. Antigen retrieval is done for 20 minutes at 90C. We use 10% normal donkey serum with 0.1% Triton-X in 1%BSA/PBS so the permeabilization is done at the same time as the blocking step. The first time it was done for 1 hour and the second time for 2 hours and neither trial was successful. There was absolutely no specific staining. Our slides are washed in PBS not TBS. My secondary antibodies work regularly with other primary antibodies.
Further optimization could be carried out such as adding a human sample in parallel to be sure that the antibody is working and trying different antigen retrieval agents, retrieval times and antibody dilutions and incubation times.
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Submitted Dec 15 2010