Eclipse e600 epifluorescence microscope

For skeletal muscle immunofluorescence, cryosections (7μm) were fixed in 4% paraformaldehyde (Merck, Darmstadt, GE), blocked for 1 hour in blocking buffer (1% BSA, BM-0150, Winkler, Santiago, CL), 1% gelatin from cold water fish skin (G7765, Sigma, St. Louis, MO, USA), 0,01% Triton X-100 (X100-1L, Sigma, MO, St. Louis, USA) in PBS, and incubated overnight at 4°C with the following antibodies: anti-fibronectin (Sigma-Aldrich, St. Louis, MO, USA), anti-collagen-I (Abcam, Cambridge, UK), anti-p-Smad3 (Cell Signaling, Danvers, MA, USA), anti-Tcf4 (Cell Signaling, Danvers, MA, USA), anti-PDGFRα (R&D Systems, Minneapolis, MN, USA) anti-laminin (Sigma-Aldrich, St. Louis, MI, USA). The corresponding Alexa Fluor 568 or 488-conjugated anti IgGs (Invitrogen, Carlsbad, CA, USA) were used as secondary antibodies. For nuclear staining, sections were incubated with 1 μg/mL Hoechst 33258 in PBS for 10 minutes [42 (link)]. Slices were then washed in water and mounted in fluorescent mounting medium (DAKO, Santa Clara, CA, USA). Sections were visualized on a Nikon Eclipse E600 epifluorescence microscope or a Nikon Eclipse C2 si confocal spectral microscope using NIS-Elements software v4.20, 32 bit.

Kidney cryosections were fixed with 4% paraformalin for 15 min at room temperature and immersed in 0.2% Triton X-100 for 10 min. After washed by TBST, slides were blocked with 10% donkey serum for 1 h, then immunostained with primary antibodies against nephrin (20R-NP002; Fitzgerald Industries International, Acton, MA), WT1 (sc192; Santa Cruz Biotechnology, Dallas, TX) at 4°C overnight. After washing, slides were incubated with CY3-conjugated, affinity-purified secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 h. Cell nuclei were stained using DAPI. Slides were observed using an Eclipse E600epi fluorescence microscope equipped with a digital camera (Nikon, Melville, NY).

Fluorescence, Phase-contrast and time-lapse microscopy (for mCherry tagged protein localisation, cell length and gliding motility studies) was carried out on either a Nikon Eclipse E600 Epifluorescence microscope or a Nikon Ti-E inverted fluorescence microscope.

To assess the degree of fibrosis, paraffin-embedded renal tissue sections were stained with Masson's trichrome stain or Sirius red stain using standard protocols11 . Stained sections were examined using an Eclipse E600 Epifluorescence microscope equipped with a digital camera (Nikon, Melville, NY, USA).

Observations were made under an Eclipse E-600 epifluorescence microscope (Nikon, Japan) using B2 filter (blue light; λ = 465–496 nm) for the rabbit anti-chicken FITC-conjugated antibody and DiOC6(3), and U2 filter (UVB light; λ = 340–380 nm) for DAPI- and AB-stained cells. All images were recorded at exactly the same time of integration using DS-Fi1 CCD camera (Nikon, Japan). Axial scans taken along representative cell files were plotted using ImageJ software. Confocal observations were made with Leica SP8 (Germany) using laser lines 405 nm (DAPI) and 553 nm (Alexa Fluor^® 555). Images were acquired with HCPL APO 63×/1.40 oil. Confocal microscopy was performed in the Laboratory of Microscopic Imaging and Specialized Biological Techniques at the Faculty of Biology and Environmental Protection, University of Lodz. The cell cycle positions (if needed) were estimated by morphometric analysis (cell length measurements) combined with the cytometric analysis of nuclear DNA (C value) after DAPI staining. All immunofluorescence analyses and Western blot assays were repeated at least twice, while other experiments were repeated several times. For statistical analysis, the intensities of immunofluorescence were measured for 50 cell wall areas in 50 (2 cell stage) and 25 (later stages) individual filaments. One-way ANOVA and Bonferroni post hoc test were applied.

Primary epicardial cells cultured on coverslips were first fixed in 4% paraformaldehyde (PFA) at room temperature for 15 min. Next, 0.25% Triton was used to permeabilize the cells for 10 min at 37 °C. Treatment with 10% goat serum for 30 min at 37 °C was used to block non-specific binding, and the cells were subsequently incubated with primary antibodies directed against Tbx18 (1:100, Abcam), ZO-1 (1:100, Abcam), HIF-1α (1:50, Abcam), Myh11 (1:300, Abcam), α-SMA (1:200, Abcam), Snail (1:100, Abcam), or Slug (1:100, Abcam) at 4 °C for 24 h. We then incubated the cells with Cy3-conjugated goat anti-rabbit IgG (CWBIO) for 45 min at 37 °C and stained the nuclei with 4′, 6-diamidino-2-phenylindole (DAPI) for 6 min at room temperature.
For frozen sectioning, mouse embryos were isolated, fixed in 4% PFA for 4–6 h at 4 °C, dehydrated using 30% sucrose for 24 h at 4 °C and then embedded in OCT. We sectioned the entire embryos into 10-μm-thick sections, incubated the sections with primary and secondary antibodies and stained the nuclei with DAPI, as described above. The stained cells and sections were photographed using a Nikon Eclipse E600 epifluorescence microscope.