I used an anti-Neu1 antibody in order to examine the localization of Neu1 in cerebellar granule precursor cells and wondered why it is not localizing to lysosomes as expected. So I used HeLa cells for positive control and there the localization is correct. Does someone know if there is a functional reason why it is different in my cell line? Or did someone else check this and has different results?
Hi Aleksandra! Could you please provide some info about the fixation / permeabilization method you are using - it might help to figure out the problem.
Hi Norbert! Cells were fixed with 4% PFA for 30 min and permeabilized 10 min with 20 ug/ml Digitonin. But the same was used for HeLa cells where Neu1 localization was fine.
HeLa cells are more "robust" than neurons. Have you tried to decrease the concentration of digitonin or incubation time and see if it improves?
No, I haven't... but the other two enzymes that are usually in complex with Neu1, beta-galactosidase and PPCA, localize to lysosomes with this approach.
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