I am trying to optimize a protocol to detect pSTAT3 on tumor cells (adherent) and see if the inhibitor will inhibit the pSTAT3 level. I have some dumb technical questions as I do not see . I culture the cells (0.5 mill in 2 ml media in 6 well plate) over night followed by treatment next day for 24 hrs. After treatment I add PFA directly to the media to make final concentration of 2% and incubate at 4C for 20 mins. Then scrape off the cells, centrifuge, wash once with PBS then add 1 ml 100% methanol to the pellet, incubate at -20C for 1 hr then wash with PBS followed by staining with pSTAT3 antibody.
I do not see any effect of inhibitor on level of pSTAT3 that made me to suspect the protocol
Do I need to wash the cells with ice cold PBS and fix with fresh 2% PFA made in PBS?
Any tweaks that might be helpful?
Do I need to serum starve the cells?
Thanks a million
I do not know what cell line or inhibitor you are using, it is possible that there is a loss of regulation in the cell line that you are using. In general, with most of these Phospho-signaling types of experiments, your briefly treat with a drug and then perform the flow. For example: " After G-CSF or IL-6/sIL-6R stimulation at 37° for 15 minutes, cells were fixed in 2% paraformaldehyde and permeabilized in 100% ice-cold methanol, as described.14 Cells were washed twice in staining buffer (DPBS pH 7.2 with 0.2% BSA and 0.09% sodium azide) and labeled for flow cytometry. All intracellular flow cytometry antibodies included pY-Stat3-PE (clone 4/P-Stat3), pS-Stat3-Alexa Fluor 488, pY-Stat5–Alexa Fluor 647, pY418-Src–Alexa Fluor 488 (clone K98-37), and total Stat3-PE (clone M59-50) antibodies, as well as fluorochrome-conjugated isotype control antibodies."
Dear Pradeep,
Is it possible that in your case the phosphorylation signals are lost due to all the staining procedure.? Maybe that is the reason why you are unable to detect any changes in pSTAT in the final read out.
For immunostaining of phosphorylated proteins I would recommend to include PhosSTOP at the fixation step.
Pls find more information at- https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Roche/Bulletin/1/phossrobul.pdf
Using phosSTOP will ensure that the phosphorylated sites will be preserved throughout the staining procedure.
Also, when I performed immunostainings in the Drosophila tissue, I used to wash the tissue with 1X PBS (at room temp) then followed by Fixative (4% PFA) containing the phosSTOP. But for your cells in vitro maybe the ice-cold PBS would be better to reduce protein degradation.
Hope this will help to improve your results.
Best wishes,
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