Tcs sp5x microscope

Sections were prepared, dewaxed, and rehydrated as for histology. After antigen retrieval (citrate buffer at 95 °C for 20 min, then slowly cooled back to RT), cross-reactivity was blocked (10 % donkey serum for 30 min). Primary antibodies were incubated at 4 °C overnight, and secondary antibodies were incubated at RT for 1 h. Sections were labelled for OC marker (integrin β3) (Barbeck et al., 2017 (link); Nakamura et al., 2007 (link)), actin (TRITC-conjugated-Phalloidin) and nuclei (DAPI). Sections were coverslipped with Mowiol® and imaged with a Leica TCS SP5X microscope.

For morphological evaluation, worms were mounted on 2% agarose pads, paralyzed with 1 mM levamisole, and visualized under a Nikon Eclipse Ti-5 fluorescence microscope with 40× or 60× magnification under Nomarski optics or fluorescence. For high-resolution images, we used a Leica TCS SP5X microscope. DAF-16 nuclear expression and MEC-4 localization in CF1139 and TU3755 animals, respectively, were quantified using ImageJ (1.46v). For accuracy in the categorization and to avoid damage due to long exposure to levamisole, animals were scored within 20 minutes after placing them on the agarose pads.

For immunofluorescence, cells were plated on glass coverslips. At 72 h post transfection, cells were fixed with 4% paraformaldehyde in PBS for 15 min, and permeabilized with 0.1% TritonX-100 in PBS for 10 min at room temperature. After blocking with 1% BSA in PBS for 45 min, the cells were incubated with the anti-γH₂AX (Dilution1:100) overnight at 4°C. The cells were then washed sequentially in PBS and incubated with Alexa Fluor 555 anti-rabbit secondary antibody for 2 h at room temperature. Finally, the nuclei of the cells were stained with 4’, 6-Diamidino-2-Phenylindole Dihydrochlorie (DAPI) for 10 min at room temperature. The stained cells were visualized on a TCS-SP5X microscope (Leica, Milton Keynes, UK)

Cells were grown on coverslips in 35 mm dishes. At 50% confluence, the cells were infected with wt CHIKV, SFV or their mutant versions harbouring substitutions mutations in nsP3 at an MOI 10, mock-infected cells were used as a control. At selected time points, the cells were fixed with 4% paraformaldehyde for 10 min and subsequently permeabilized with 0.5% Triton X-100 for 3 min. Cells were washed with PBS, blocked with 5% horse serum in PBS, and stained for 1 h with primary antibodies against SFV, CHIKV nsP3 (both rabbit, in-house), CD2AP (mouse (B4) sc-25272; rabbit (H290) sc-9137), SH3KBP1 (mouse sc-166862, Santa Cruz Biotechnologies) and/or dsRNA (mouse J2, Scicons, Szirák, Hungary). Incubation with primary antibody was followed by incubation with secondary anti-mouse or anti-rabbit antibodies conjugated to Alexa Fluor555 or Alexa Fluor488 (Thermo Fisher Scientific). Draq5 (BioStatus, Loughborough, UK) was used to counterstain nuclei. Washed coverslips were mounted in mounting medium and images were obtained and analysed using a LSM710 confocal microscope (Zeiss, Oberkochen, Germany) or TCS SP5 X microscope (Leica, Wetzlar, Germany) equipped with a super continuum pulsed white laser. Images were processed using Photoshop (Adobe Systems Incorporated, San Jose, CA, USA).