HEK 293 cells were transfected with GFP tagged plasmid in earlier before doing immunostaining on cover slips. Under flurescence microscope I saw GFP continuously. But, during intracellular staining I used 4% PFA in 1X PBS for o/n at 4 degrees for fixation and subsequently used all other antibodies according to the protocol. Unfortunately, on confocal microscope surprisingly I didn't see GFP on cover slips. Why? Secondary Ab was tagged with Alexa 555 and 594. Both are red channels. Could you please share your experiences. How can I improve my protocol?
In our lab we usually use 3% PFA in PBS and we fix different type of cells on glass slides for about 20 min at room temperature. I think 4% PFA o/n fixation is too long and only used for tissue sections. After fixation, we remove the PFA and store the glass slides in 1xPBS at 4° C until IF staining.
How do you permeabilize your cells, do you use triton-X? Permeabilization can also have an impact on your GFP-signal intensity. I would recommend using 0.1% saponin in PBS for permeabilization, 15 min at RT should be sufficient. This is a much milder way to permeabilize your cells in comparison to triton-X.
Thanks a lot @Alexander Gluschko. Yes, I use Triton X-100. Next time I will keep in my mind your valuable suggestions.
The fluorescence of GFP depends on the integrity of the 3-D structure of the protein. It is thus not surprising that it should be destroyed by PFA fixation.
You may still use anti GFP antibody with Alexa488 or even more blue shifted fluorophore
Dear Adam Figarski thanks for your great suggestion. I'll try it at next time.
Fixation only for 20 minutes at room temperature, then permeabilize! why overnight? This works very well for IF
Hi Obaidur,
We have fixed with 4% PFA 15 mins at room temperature and used Alexa fluor 488 for GFP in astrocytes. It works absolutely fine. You could also try this.
All the best!
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