I am trying to co-stain for GFP and a ChAT marker in the brain of a mouse model that has GFP-expressing neurons. Typically I get a strong fluorescent signal when I stain only for GFP (photo 1), but when combining with the ChAT marker the fluorescence seems to disappear (photo 2, only showing the GFP channel).
I am blocking in 1% BSA 0.5% Triton X-100 and use this to prepare the following primary antibodies: rabbit anti-ChAT (1:500 dilution) and goat anti-GFP (1:1000 dilution). I incubate overnight at 4C. The secondary antibodies I am using are donkey anti-goat 488 and donkey anti-rabbit 594, also prepared in 1% BSA 0.5% Triton X-100 blocking solution.
Does anybody know why this is happening?
Photo 1 - Staining with GFP only. CA1 region of the hippocampus.
Photo 2 - Co-staining of GFP with ChAT, only showing the GFP channel, in the dentate gyrus.
My previous work has confirmed that GFP is present in both the CA1 region and the DG in this model.
Good evening,
I would be happy to assist where I can, to further diagnose the problem I'll need to have a few more pieces of data which I’ve listed below if possible.
1. Images of the ChAT stain alone and co-stained with GFP.
2. what is your imaging timing e.g., is this imaged sequentially (single channel at a time) or in parallel (multiplex)?
3. Do you have a negative label controls to compare your results with possible background tissue autofluorescence?
4. which stain is being applied first?
Hi Zackery,
1. I have attached the files below.
2. Each channel is imaged separately (red - green - blue)
3. File attached below.
4. I'm using free-floating sections and therefore combine the primary antibodies in the blocking solution, i.e. they're applied simultaneously.
Thanks for your help!
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