Hi, I am staining the TLR4 membrane receptor on ARPE-19 cells.
I did not use any permeabilization, however, every time I can see some signals from inside of the cells. I think it is Golgi apparatus.
Do I need to change the primary antibody? Thanks
To be sure it is from inside the cell you should use confocal microscopy. You can see signals from inside the cell, if the internalisation pathways are intact. You cn try to add some drugs to inhibit internalisation. For TLR4 i is likely that what you see inside the cell corresponds to golgi (newly processed proteins)
Though there are intracellualar TLR4 pools, your antibody should not be entering the cells. Internalization slows below 22 oC. Some reagent, detergent or osmolar condition in your staining procedure is causing permeabilization.
Have you fix your sample prior to immunostain it ? If so, what fixing reagent did you used ? methanol for instance induces membrane permeabilzation. I second Jose A Garcia-Sanz regarding TLR4 localization in the Golgi.
Hi Alexandre,
thanks for your kind help! Methanol does induce the membrane permeabilization, and I used 4% PFA to fix the cells, however, it also cause permeabilization of the cell membrane. With confocal microscopy, I could see the intracellular TLR4 everywhere. Now I am still testing other methods to fix the cells.
Best,
Sai
first rather check your signal and your technical details. 4% PFA does not permeabilize cells: i had never heard that before. stock of FA solutions contains some methanol as stabilizer, but you mentioned PFA.
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