Anti ck18 antibody

For immunofluorescence staining, mpPHH cultures were fixed and stained using as primary antibodies a rabbit polyclonal anti-NuMa antibody (catalog no. ab97585; Abcam) and a mouse monoclonal anti-CK18 antibody (catalog no. ab82254; Abcam). AlexaFluor 488 and 594 (Thermo Fisher) of the corresponding species were used as secondary antibodies. Nuclei were stained with DAPI. Cells were imaged using a Nikon Eclipse TE300 fluorescent microscope and processed using ImageJ.

The MCs were seeded onto glass slides and once a monolayer was formed, the culture medium was discarded and the cells were washed with PBS. After fixing with 4% paraformaldehyde for 15 min at room temperature, the MCs were rinsed thrice with PBS, permeabilized in 0.3% Triton x-100 (Sigma, USA) for 20 min, and washed again. The samples were blocked with 5% BSA (bovine serum albumin) for 10 min, and incubated overnight with anti-CK18 antibody (1:500; Abcam, USA). After washing thrice with PBS, the cells were incubated with anti-rabbit IgG (diluted 1:100 to 1:200 with 1% BSA) for 30 min. The slides were suitably processed, mounted and observed under a fluorescence electron microscope.