Lab tekii chamber slide

The human U-251 astrocytoma cell line (U-251MG, RRID:CVCL 0021) was cultured in DMEM high glucose medium with 10% heat-inactivated foetal bovine serum (FBS) and penicillin–streptomycin. Cells seeded on Nunc Lab-TekII Chamber Slide™ were treated overnight with 1 µg/ml tunicamycin (Sigma) or vehicle before being fixed 10 min with 2% paraformaldehyde in PBS and further processed for immunohistofluorescence with rabbit anti-Cter Kir4.1_356-375 and mouse or human HR anti-e1 serum followed by the donkey AF488-coupled F(ab’)2 anti-rabbit IgG and AF594-coupled F(ab’)2 anti-mouse or anti-human IgGs. For competition experiments (with e1-preadsorbed sera), mouse (1:100) or HR (1:50) anti-e1 sera were incubated 36 h at 4°C with e1 peptide at 20 µg/ml in PBS supplemented with 2% BSA, centrifuged (14 000 g) for 30 min to remove antibody complexes before being applied to tissue sections at desired concentration. Slides were coverslipped with Prolong Gold DAPI mounting medium before taking pictures at ×40 objective under the fluorescent microscope at fixed exposure time.

10⁴ cells were seeded onto an 8-well Lab-TekII chamber slide, fixed in 3.7% formaldehyde solution (Sigma), and permeabilized in 0.1% Triton 100X (Sigma), PBS buffer at room temperature for 10 min. The cells were then washed 3 times with PBS and incubated for 1 h with 10% horse serum in PBS blocking solution. The cells were incubated overnight at 4°C with murine monoclonal anti-E-cadherin clone 36, (Becton Dickinson), anti-β-catenin clone 14 (Becton Dickinson), anti-fibronectin clone 10 (Becton Dickinson), anti-vimentin clone V9 (Dako), or with a polyclonal rabbit anti-HA Y11 (Santa-Cruz) primary antibody, washed in PBS, and then incubated for 1 h at room temperature with a goat anti-mouse Alexa Fluor 533 secondary antibody (A21422, Invitrogen) or with a mouse anti-rabbit Alexa Fluor 555 secondary antidody (A21428, Invitrogen). After extensive washes in PBS, the nuclei were stained with 5 mg/ml Hoechst for 10 min and mounted with Fluoromount-G (SouthernBiotech). All matched samples were photographed with an immunofluorescence microscope (Leica) and identical exposure times.