When I stained the presynaptic sites of NMJs in the mouse lumbrical muscle, I observed significant background in my IFI.
I fixed them with 0.5% formaldehyde overnight at room temperature. Currently, I suspect my formaldehyde solution, as it is quite old. Next, I blocked with PBS + 0.5% Triton X-100 (PBST) + 4% BSA overnight at 4°C. Then, I incubated with primary antibodies (SV2/2H3 from DSHB, both at 1/250 dilution) raised in mouse overnight at 4°C. Following this, I incubated with secondary antibody (anti-mouse Cy2) raised in goat overnight at 4°C. Both antibodies were diluted in the blocking solution.
Any comments, suggestions, or changes I can make are greatly welcomed.
Lumb1 = negative control without primary antibody Lumb2 = Presynaptic and AChR cluster Yellow: Presynaptic (SV2 and 2H3) Magenta: AChR
(Both images were captured using an epifluorescence microscope).
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