Eclipse 50i epifluorescence microscope

For immunofluorescence (IF) staining, HeLa cells were seeded on glass coverslips, and 24 h later, they were fixed for 15 min at room temperature (RT) in 2% formaldehyde in PBS. After permeabilization in 0.2% Triton X-100 solution and washing in PBS, the cells were incubated in 3% bovine serum albumin (BSA) for 1 h and, subsequently, with primary antibodies overnight at 4 °C. After several washes in PBS, the cells were incubated with secondary antibodies for 1 h at RT. After washing in PBS, the coverslips were counterstained with DAPI and mounted onto slides with ProLong™ Diamond Antifade Mountant (Invitrogen). Preparations were analyzed using a computer-controlled Nikon Eclipse 50i epifluorescence microscope equipped with a CCD camera.
For quantitative analysis of immunofluorescence images, a dedicated tool of Fiji software was used. In brief, a mask was automatically created on α-tubulin signal and manually adjusted by setting up a proper threshold to eliminate the noise. The mask was converted in a selection to apply on remodeler signal which needed to quantify. A set of 5 random background areas from the same image were used to normalize the integrated intensity of the specific signal.

For IFAT, 1 × 10³ SHK-1 cells were seeded on coverslips (n = 3), treated, and infected as was described above. Then, cells were fixed with 4% paraformaldehyde in PBS for 30 min, permeabilized with 0.1% saponin in PBS, and blocked with 0.1% saponin in PBS plus 3% BSA for 30 min. Intracellular P. salmonis was detected by incubation with specific antibodies anti-P. salmonis (ANGO, USA) at 1:200 dilution for 1 h at room temperature. After three washes with PBS, cells were incubated with secondary anti-mouse FITC-conjugated antibody (Thermo Fisher Scientific, USA) at 1:200 dilution for 1 h and with phalloidin-Alexa 636 (Thermo Fisher Scientifi) at 1:200 dilution for 15 min to detect polymerized actin; and DAPI (4′,6-diamidino-2-phenylindole, Thermo Fisher Scientific, 1:200 dilution) for 15 min to detect DNA. Preparations were observed in the Eclipse 50i epifluorescence microscope (Nikon, Japan) at 100 × magnification under oil immersion [23 (link)]. The images obtained were processed with the Fiji Image J program (GNU General Public License).

Sediment samples were fixed in 3% formaldehyde for 3 h at 4°C, washed with 1x phosphate-buffered saline (PBS) and stored in ethanol-PBS (1:1) at -20°C. Samples were diluted, four times ultrasonicated on ice at 20% intensity, 20 cycles, 30 s (Bandelin Sonopuls HD200). An aliquot was filtered on a 0.22 μm pore size polycarbonate filter. Filter sections were embedded in Citifluor:Vectashield (4:1) mounting medium containing 50 μg ml^-1 DAPI. Microscopy was done with a Nikon eclipse 50i epifluorescence microscope.

Immunolocalization experiments to Drosophila polytene chromosomes were according to Deuring et al.25 (link). Anti-GFP rabbit polyclonal antibodies was used at 1:100 dilution, while anti-CFDP1 mouse monocolonal antibodies (Abnova, H00010428-M4, clone 5B7) at 1:50. anti-H2A.V or anti-H2A (Millipore) antibodies were used at 1:100 dilution. After three washes in PBS, they were incubated with secondary antibodies Alexa Fluor 555 goat anti-rabbit IgG (1:300) for 1 h at room temperature and DAPI were used to DNA staining. Polytene chromosome preparations were analyzed by using a computer-controlled Nikon Eclipse 50i epifluorescence microscope equipped with a CCD camera. Measurements of polytene chromosome fluorescence levels of H2A.V and H2A were performed using the ImageJ software. For each histone, 25 chromosome figures were analysed in w, elav-GAL4^[w+]/w; UAS-Cfdp1^[w+]/+ larvae and in w/w UAS-Cfdp1^[w+]/UAS-Cfdp1^[w+] control larvae.

We examined six coronal sections per rat assayed by double immunofluorescence against MCHR-1 and serotonin obtained from three rats. In each coronal section, five 20x microphotographs were taken along the dorso-ventral axis of the serotonergic (central) region of the MnR, with a 20x objective lens on a Moticam Pro 282B camera (Motic, Hong Kong, China), coupled to an Eclipse 50i epifluorescence microscope (Nikon, Tokio, Japan). Photoshop and Image J softwares were used to quantify the number of serotonergic neurons in each microphotograph, as well as the number of MCHR-1 positive signals in structures recognized as primary cilia in our previous study^{19 (link)}. The number of counted cells were averaged per animal; thereafter, a grand average was performed for all the animals. The values are presented as mean±S.E.M. (standard error of the mean).

AGS and MKN45 cell lines were seeded (50,000 cells) on glass coverslips in 24-well plates, serum-deprived 24 h later and treated with LIF at different time intervals (0.5, 2, 5, 24 and/or 48 h). Cells were then fixed with 3% paraformaldehyde solution in cytoskeletal buffer prior to immunofluorescence staining. After permeabilisation with 0.1% Triton X-100 solution for 1 min and blocking in 1%-BSA 2%-FBS in tris-buffered saline (TBS) solution, cells were stepwise incubated in corresponding primary antibodies for 1 h, washed 3 times in TBS and left for 30 min in secondary antibody coupled with 1:350 Alexa Fluor^® 488 goat anti-rabbit or anti-mouse IgG antibodies, 1:250 Alexa Fluor^® 647-coupled Phalloidin and 50 mg/mL 4′-6-diamino-phenyl-indol (DAPI) (all from ThermoFisher Scientific, Villebon sur Yvette, France). Primary antibodies used were 1:100 rabbit anti-YAP and 1:100 mouse anti-TAZ E5P2N (cat.4912 and cat.71192, respectively; Cell Signalling Technology, Saint-Cyr-L’École, France). Images were taken using an Eclipse 50i epi-fluorescence microscope (Nikon, Champigny sur Marne, France) with the NIS-BR acquisition software and a ×40 (numerical aperture, 1.3) oil immersion objective. Nuclear YAP/TAZ count and mean grey value measurements were carried out using the ImageJ 1.52p software (National Institutes of Health, Rockville Pike, Bethesda, MA, USA) [44 (link)].