Imic digital microscope

Cells were seeded in ibidi μ-slide eight-well plates. After respective treatments, cells were washed and fixed with 4% paraformaldehyde (PFA) and 100% ice-cold methanol. After permeabilization with 0.4% Triton X-100, quenching with 100 nM glycine, and blocking with 10% FCS, anti-C2IN serum, and subsequently fluorescence labeled secondary antibody were added. Images were obtained using iMic digital microscope (FEI Munich) and Live Acquisition 2.6 software (FEI Munich) and were processed with ImageJ software (National Institutes of Health, Bethesda).

Cells were incubated as indicated at 37 °C in 5% CO₂. Afterwards, cells were washed twice and fixed (4% PFA, Sigma-Aldrich, St. Louis, MO, USA). For MHC class II staining, cells were incubated with 2% BSA in PBS and labeled with anti-HLA-DR antibody (1:200, L243, Leinco, St. Louis, MO, USA) for 30 min at RT. After three washing steps with PBS, MHC class II was detected by Cy5-conjugated goat anti-mouse antibody (1:250, Dianova, Hamburg, Germany). If indicated, cell nuclei were stained either with DAPI (1:200, Sigma-Aldrich, St. Louis, MO, USA) diluted in 1% BSA and 0.1% Triton X-100 in PBS, or with 5 µg/mL Hoechst33342 in PBS for 10 min at RT. Confocal images were acquired by using the inverted laser scanning confocal microscope LSM 710 (Zeiss, Oberkochen, Germany). Epifluorescence microscopic images were recorded using the iMIC digital microscope (FEI, Munich, Germany). Images were processed using ImageJ software (v1.51n, National Institute of Health, Bethesda, MD, USA).

Cells were seeded in 8-well plates (ibidi, Martinsried, Planegg, Germany). After treatment with toxin components, cells were washed with PBS and fixed with PFA for 15 min. After washing with PBS, cells were permeabilized with Triton X-100, and autofluorescence was quenched with glycine. Then, cells were blocked in 5% milk powder in PBST and incubated with primary antibodies (mouse anti-α-tubulin (Santa Cruz Biotechnology, Dallas, TX, USA), goat anti-EEA1 (early endosome marker, Sicgen, Cantanhede, Portugal)), and after washing with PBST with secondary antibodies (goat anti-mouse-568 (Thermo Fisher Scientific, Waltham, MA, USA), mouse anti-goat-647 (Santa Cruz Biotechnology)). F-actin was stained with Phalloidin-FITC (Merck, Sigma-Alrich) and nuclei with Hoechst 33,342 (Thermo Fisher Scientific). Images were obtained with an iMic Digital Microscope using the Live Acquisition 2.6 software (FEI, Munich, Germany) and were processed with ImageJ.