puncta staining problem - (Jul/18/2006 )

Hi,

I am trying to do some immunocytostaining specific to membrane proteins in neurons. I've tried two way to apply my primary antibody: 1, add antibody to culture media and RT for 10 min; 2, PFA fix but not permiblize cells, and add antibody RT 1 hr. Then, add secondary antibody.....
However, I got bright staining within whole dendrites. No puncta at all. I've try to the antibody that works well in other lab. But I still cannot get puncta staining. It seems something wrong with my fixation. But I use PFA 4% in PBS,pH7.4. warm up to 37C before use. And wash cells with PBS before add PFA. Then fix at RT for 10min. I am so frustrated, I do not know what happens to my PFA fixation or there are some tricky I do not know. Do you face the same problem? Could some guys help me? Thanks!


SkyS

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R u sure that the staining has to b punctate, may b the protein is widely spread that it gives a confluent staining.

Try changing blocking agents like NGS instead of FBS or vice versa.

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Sure, it should be puncta after staining. It is PSD-95 and the antibody works well in other lab. What I am not unsure is whether 4%PFA can also permiblize cell. I did not block at all, after primary, I added secondary directly.

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Try blocking ur cells with 5% FBS. Then add primary and later secondary.

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I finally decreases my second antibody concentration, and it works. Thanks a lot all the same.

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Hi,

I'm also trying to stain for PSD95 on my primary neuronal culture. For some reason, I can not detect signal at all. I'm also expecting puncta staining, but it does not seem to work well. My neurons are plated at a density that synapse and denrities have form, etc. Can anyone help me with the protocol please? I fix in 3.7% formaldehyde for 10 min, then block with BSA, dilute primary in BSA overnight and secondary stain for 1hr in BSA.


Please help. Thank you.

--Jessica

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