Has anybody tried to stain samples for imaging yet? I want to try and stain my cleared mouse brain, but don't want to spill my AB's optimizing this. Do any of you have tips regarding staining of thick slides of mouse brain (5 mm slice)?
Does anyone have any results of staining attempts that they would be willing to share? Has anyone gotten results as well as the authors on very thick tissue pieces or should I try and work with <1mm sections?
The clarified sample(500um) was incubated at 37℃ for 1 day in 0.1%TritonX-100,primary antibody(anti-tublin ,T6793,dilution-1:100)and 1M sodium borate buffer solution ,pH8.5,followed by wash 37℃ for 1day with 0.1%TritonX-100 and 1M sodium borate buffer solution ,pH8.5.
The tissue was then incubated at 37℃ for 1 day in 0.1%TritonX-100,secondary antibody(cy3-goat-antimouse,dilution-1:100)and 1M sodium borate buffer solution ,pH8.5,followed by wash 37℃ for 24h with 0.1%TritonX-100 and 1M sodium borate buffer solution ,pH8.5. And before imaging ,the sample was incubated in FocusClear(300ul) for 24h.
The volume of the diluted staining dye was about 1500ul in a tube(2ml).And when I incubated my sample in the secondary antibody,I also added DAPI in the system(final conc:2ug/ml).But it seemed that the backgound was also too high to recoginzed the nucleus.
I got a huge autofluorescence background both in red and green channel while imaging ETC cleared, and Ab-stained mouse pancreas. To reduce the tissue autofluorescence I even bleached the tissue for 16h in a mixture of MeOH:DMSO:H2O2. The primary and secondary antibodies were diluted in 10% serum in TBST buffer with 5% DMSO. Unfortunatley I could not see any specific staining. Has anybody experienced similar problem with any other mouse tissue ? Pl. share your experience or how to overcome that ? ...Thanks!
You should complete staining after incubating your samples a couple days in PBST. Don't put them in FocusClear until right before you're ready to image (0-1 days).
I'm afraid my attempt failed. I'm going to repeat when I have a chance. This time, I think I'll stick to passive clearing and try a smaller piece of tissue (I tried a kidney half).