Phospho myosin light chain 2 ser19 antibody

Cells were fixed with a solution of 4% paraformaldehyde in 1× PBS for 15 min and permeabilized with 0.2% Triton X-100 for 20 min. The cells were then blocked with 2% bovine serum albumin (BSA) in 1× PBS (PBS-2% BSA) at room temperature for 1 h, incubated with primary antibodies for Paxillin (Recombinant Anti-Paxillin antibody [Y113], Abcam ab32084; 1:250 dilution) and Myosin (Phospho-Myosin Light Chain 2 [Ser19] Antibody, Cell Signaling Technologies [#3671]; 1:250 dilution) in PBS-2% BSA for 48 h at 4°C. The sample was incubated for 48 h at 4°C for secondary antibodies Alexa Fluor 647 (Donkey anti-Rabbit, Abcam ab 150075, 1:500 dilution) and Alexa Fluor 555 (Anti-mouse IgG (H+L), F(ab’)2 Fragment, 1:500 dilution). Phalloidin staining was performed with Alexa Fluor 488 Phalloidin (Life Technologies; 1:200) diluted in PBS with 2% BSA for 48 h at 4°C. Images were taken with 2× (air), 40×, and 60× oil-immersion objective. Analysis was performed with ImageJ and Imaris (Bitplane).

For immunostaining, cells were fixed in ice-cold 3.7% paraformaldehyde in intracellular buffer (140 mM KCl, 1 mM MgCl₂, 2 mM EGTA, 320 mM sucrose, and 20 mM HEPES with pH 7.5) for 45 min on ice. For imaging fMLP internalization, cells were washed with acid buffer (500 mM Glycine in mHBSS, pH = 2.7) prior to fixation. After washing and permeabilization with 1% Triton X-100, cells were then blocked in intracellular buffer containing 1% bovine serum albumin for 1 hr, before incubating with primary antibody overnight at 4 °C. On the second day, after washed six times with PBS, samples were incubated with secondary antibody along with rhodamine-phalloidin (PHDR1, Cytoskeleton, CO) and DAPI (D9542, Sigma-Aldrich, MO) for 3 hours at room temperature at manufacturer's recommended concentrations. Cells were imaged after washing with PBS.
In immunostaining experiments, we have used the following antibodies: AF647 mouse – α-human fMLP receptor (565623, BD Biosciences, CA), phospho-myosin light chain2 (Ser19) antibody (#3675, Cell Signaling Technology, MA), α-tubulin antibody (#2144, Cell Signaling Technology, MA), clathrin antibody [X22] (ab2731, Abcam, MA), β-arrestin-1/2 antibody (sc-74591, Santa Cruz Biotechnology, TX).