Carl apotome axiovert 200 m fluorescence microscope

Cells were cultured on glass coverslips in 6-well plates to at least 80% confluence and fixed in ice-cold methanol for 20 min for both proximity ligation assays (PLA) E-cadherin/b-catenin and E-cadherin/p120. PLA was performed using Duolink Detection kit (Olink Bioscience, Sweden), according to the manufacturer’s instructions for Duolink Blocking solution and Detection protocol. Briefly, slides were blocked, incubated with antibodies directed against E-cadherin cytoplasmic domain (610,182, BD Biosciences or Clone 24E10, #3195, Cell Signaling, USA), b-catenin (C2206, Sigma-Aldrich, USA) and p120 (610,134, BD Biosciences, USA), followed by incubation with the secondary PLA probes (anti-mouse Minus and anti-rabbit Plus) conjugated to unique oligonucleotides. Amplification template oligonucleotides were hybridized to pairs of PLA probe and circularized by ligation. Rolling circle amplification was performed and detection of amplified DNA was possible by addition of complementary oligonucleotides labeled with Cy3 fluorophore. Coverslips were mounted on Vectashield with DAPI (Vector Laboratories, USA). Images were acquired on a Carl Zeiss Apotome Axiovert 200 M Fluorescence Microscope (× 20 and × 40 objectives; Carl Zeiss, Germany) with an Axiocam HRm camera and processed with the Zeiss Axion Vision 4.8 software. Quantification of PLA signals was achieved using BlobFinder V3.2.42.

Cells were cultured to confluent monolayers on glass coverslips and fixed in ice-cold methanol for 20 min, except for S100P stained cells, which were fixed with 4% paraformaldehyde for 20 min. Following a 10 min PBS wash, cells fixed with paraformaldehyde were incubated in NH₄Cl 50 mM for 10 min, and permeabilized with 0.1% Triton X-100 in PBS for 5 min, at room temperature. Cells were blocked with 3% BSA in PBS and stained with primary antibodies, rabbit anti-S100P (ab133554, Abcam, UK) and mouse anti-E-cadherin (610,182, BD Biosciences, USA), followed by a 1 h incubation in the dark with Alexa 488 or Alexa 594-conjugated secondary IgG (Invitrogen, Thermo Fisher Scientific, USA). Coverslips were mounted with Vectashield with DAPI (Vector Laboratories, USA) and images acquired on a Carl Zeiss Apotome Axiovert 200 M Fluorescence Microscope (× 20 and × 40 objectives; Carl Zeiss, Germany) with an Axiocam HRm camera. Images were processed with the Zeiss Axion Vision 4.8 software.

Bacteria images were acquired with a Carl Zeiss Apotome Axiovert 200M Fluorescence Microscope (Carl Zeiss, Jena, Germany). Images were taken with an Axiocam HRm camera and processed with Zeiss Axion Vision 4.8 software. All the experiments were performed in triplicate.
AGS (gastric adenocarcinoma) cells were analyzed on an inverted epi-fluorescence microscope, (Axiovert 200M, Zeiss,Germany). Images were acquired with a Leica TCP SP2 AOBS camera and processed LAS AF using Lite software (Leica Microsystems CMS GmbH).