why EEA1(early endosome antiboy) stained to nuclei? - (Jun/18/2014 )

Hello guys~

i attached the picture and have a some problem.

i stained EEA1(early endosome marker). however there are many spots in the nuclei. it shouldn't be observed i think.

is it normal? or should i change some protocol?

this is my short protocol

used -20 degree methanol for fixation and permeablization 10minute.

blocked with 3%BSA/PBS 30minute RT

stained EEA1 diluted in 3%BSA/PBS 1hour RT with low speed shaking.

wash 3times PBS confocal microscopy

please reply for my topic for my work. HHHHELP Me~~~

It is not uncommon to observe some EEA1 in "spots" in the nuclei (I see it quite often). However, as far as I could see in the image, the majority of the EEA1 is there rather than in the cytoplasm - THAT is not normal.

My protocol is a bit different, for fixation I use 4% PFA (20 min, RT) and for permeabilization 0,5% Tween-20 in PBS (20 min, 37°C). Washing 3 times with PBS confocal microscopy between each step is required. Usually, I don't use any type of shaking when staining cells, but that's not important in this case.

I gather that you use direct IF, so specificity should not be a problem. If not - check cross-reactivity. Also, when incubating too long with secondary antibody, the non-specific staining is more likely to happen.

What are the cells in question? Are they usually adherent or did you use cytospin to adhere them? Some manipulations with the cells before the staining process can cause them to change their subcellular structures and their position. 

Did you use confocal microscope to take that image? If not, you may have observed the EEA1 that is in cytoplasm ABOVE the cell nucleus.