I grow cells in 8-well plates, then use receptor primary antibodies. This time I used a secondary antibody that we had, which was rhodamine conjugated. One of the wells showed a bright yellow staining (cytoplasm), while the others didn't show much staining due to low concentration of the secondary antibody. If it worked as planned, the cells should have been red in the cytoplasm. I also used Hoechst (nuclear stain) during the secondary antibody incubation.
Hi Brandon few suggestions that may help to figure out what is happening with your stain
Make sure you have proper controls both positive and negative which will help to differentiate between specific and non-specific stain.
Washing in between antibody incubations to remove non specific binding
Make sure you have the proper light filters in the microscope
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