hi, everyone
Originally I use FITC 2nd antibody to label overexpressed protein while Alexa 568 to label a lysosome marker LAMP2, but the result was not good. Lysosome marker required enhancement which may contribute false signals, and signals of FITC labels were too strong that colocalization was hard to define when two images were merged.
Therefore I switch the labels and hope that FITC to label the relatively weak LAMP2 could give me better signals. Yes, it turned much stronger but it also raise my doubt.
The location of FITC-labelled "LAMP2" perfectly match the location of Alexa 568-labelled overexpressed protein.
So I checked the spectra of each 2nd antibody and found that FITC excitation wavelength 499nm could excite Alexa 568 at around 10%. Even though there is only 10%, it looks very likely that the "enhanced LAMP2" was actually the ghost of Alexa 568-labelled overexpressed protein.
Has anyone also encounter this situation before?
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