I have stained WT HCT116 cells with Gapdh (green fluorescence). Fixed the cells with 4% paraformaldehyde. Washed with 1X PBST. Blocked with 1% goat serum/ BSA. Incubated with primary Ab overnight. Secondary Ab for 1hr.
While I should see the Gapdh signal only in the cytoplasm, I am seeing this unusual unspecific signal in the nucleus. I have tried changing the antibody, fixation time, antibody conc, fixation reagent(used methanol) but still nothing changed much.
1x PBST is not a blocking agent. What protein did you block with?
hi Dhanalakshmi Jothi
I would like to know, have you stained the nucleus with DAPI since it is not mentioned in the steps you provided?
Very Important, PBST is not a blocking agent.
Then, Is this image is generated by allocating color directly to the cells, have you removed the background stain? I mean in ImageJ, there is the background subtraction option, it helps to remove background staining.
Regards
Saurabh
Hello Dhanalakshmi. How are you doing today?
Hum, analyzing the picture, this seems to be a big problem in your immunofluorescence assay. Well, check the specificity of your antibody first, if there is no cross reaction with other molecules. May be this could be the problem.
You already tested change the incubation period, antibody concentration, fixative reagent , among other things, and you still have the same problem. Also, try add some negative control without primary antibody and check the blocking steps, maybe the blocking period is not enough or the blocking reagent is not working. If you do that and still have problem maybe is not the antibody. In addition, as aforementioned, you need to add some nucleus stain such as DAPI or Propidium Iodide.
If you are continuing have this problem, maybe you have to consider that there is GAPDH in the nucleus. As a housekeeping protein, its expression is considered high. GAPDH translocate to the nucleus in some conditions. You will find more about in these papers 10.1016/j.biocel.2010.06.014 and 10.1016/j.cellsig.2010.08.003
My best regards and good luck in your research.
First, blocking with 3% BSA in PBS for 30 min at RT may solve this problem. After blocking one slide please incubate it with the secondary antibody and check if it is negative. If not, those nuclei might have autofluorescence. Please check the sample using fluorescent microscope just after fixation and washing with PBS . If you see positive staining that will be an evidence for their autofluorescence. If so, you cannot stain the cells with fluorescent reagent. You may choose non-fluorescent DAB staining using peroxidase conjugated 2nd antibodies.
Saurabh Mandal Hello Saurab, Thank you for the reply. Sry for the typo error. I washed with 1X PBS and blocked with 1% goat serum. I haven't tried background subtraction. I'll check this and see if it works
Túlio Teruo Yoshinaga Hi Tulio, Thank for your comments. I'd try out the negative ctrl w/o primary Ab and see if it works. While I did the DAPI staining and it works pretty good.
Tomoko Nakai Hi Tomoko, Tq for the suggestion. I'll check if the nuclei has autoflorescence.
What was the concentration of DAPI in your staining?
DAPI tends to bleedover in Green channel. I would recommend doing a no DAPI staining and a staining using other nuclear dye (PI or some other dye). If you still nuclear localization of GAPDH then the conditions might favor its nuclear translocation.
In addition to the solutions mentioned above, try to increase the concentration of the blocking agent. if using goat serum, I would go up to 5-10%. if using BSA, I would try 3-5%.
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