I need to do TLR4 staining on cell membrane. Every time I did it without any permeabilization, TLR4 in cytoplasm has also been stained. I have already tried PFA(4%) fixation with different fixation time(5 min, 10 min, 15min) as well as different primary antibody concentration(1:50-1:10000), and different antibody incubation temperature(37°C,25°C,4°C), however, TLR4 antibody went always into cytoplasm? Low temperature incubation of antibody worked a little bit, but there were still a lot of signals from cytoplasm. Does anyone have any idea about this? thanks
As you see the website of the antibody for TLR4 (Abcam), the staining pattern seems to be distribute for both cellular membrane and cytosol. One thing I can advise is to decrease the signal intensity when you take a picture with confocal microscopy. When I obtained F-IHC data with microscopy, the cytosolic staining of EpCAM, one of the cellular membrane proteins, was problematic. That is why I took a picture with less intensity.
http://www.abcam.com/tlr4-antibody-ab13556.html
Hi! Maybe the PFA concentration is too high, 4% can destabilize membrane lipids, even if you use methanol-free solution of PFA. I usually use 1-2% PFA when I want to make a membrane staining.
On the other hand, your cells might not express cell surface TLR4, as we and others showed in case of endothelial cells (PMID: 21290470, 15132988).
Good luck!
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