Dm irb confocal microscope

Kidney sections were stained with anti-phosphorylated EGFR (1:200; Dako Agilent, Santa Clara, CA, USA), Alexa Fluor 488 conjugated goat anti-mouse secondary antibodies (1:300; Invitrogen, Waltham, MA, USA), and examined using a Leica DM-IRB confocal microscope.

Immunofluorescence studies were assessed in VSMCs seeded in 24-well Multidish over glass coverslips (Cultek, Madrid, Spain). Once experiments were done, cells were fixed in 4% PFA, treated with 0.1% Triton-X100 and blocked with 4% BSA in TBS. Afterwards, cells were incubated with p-SMAD2 ([1/200]; #3108, Cell signaling, MA, USA) and p-SMAD4 antibodies ([1/200]; sc7966; Santa Cruz Biotechnology, Heidelberg, Germany) overnight, followed by 1 h of incubation with AlexaFluor^®488 conjugated secondary antibody (1/300; Invitrogen, Life Technologies, Philadelphia, PA, USA). DAPI was used as nuclear counterstaining (Sigma Chemical). Negative control was also performed in absence of primary antibody (data not shown) in order to verify specificity of the immunostaining. Finally, samples were mounted in ProlongGoldTM (Invitrogen, Life Technologies) and visualized in a Leica DM-IRB confocal microscope.

In paraffin-embedded kidney sections (3 µm), antigens were retrieved by the PTlink link system. After the slides were blocked with 10% BSA and 10% FBS for 1 h, they were incubated with anti-SOX9 (1/200; Millipore) or anti-type IV collagen (1:1000; Abcam) for 1 h, followed by an AlexaFluor^® 488 conjugated rabbit anti-mouse secondary antibody (1/200; Invitrogen); AlexaFluor^® 633 conjugated rabbit anti-mouse secondary antibody (1/200; Invitrogen) or AlexaFluor^® 568 conjugated rabbit anti-mouse secondary antibody (1/200; Invitrogen) for 1 h. The absence of a primary antibody was used as a negative control. Samples were mounted in prolong gold (Thermo Fisher Scientific; Waltham, MA, USA) and examined using a Leica DM-IRB confocal microscope.

Paraffin-embedded kidney sections (3 μm) were submitted for antigen retrieval. After the slides were blocked with 10% BSA and 10% FBS for 1 h, they were incubated with FGF23 primary antibody (1/200) for 1 h, followed by a AlexaFluorTM 594 conjugated secondary antibody (1/200; Invitrogen, Waltham, MA, USA) for 1 h. The absence of primary antibody was used as negative control. Samples were mounted in moviol and examined using a Leica DM-IRB confocal microscope.

Cell death detection by TUNEL assay was performed following the manufacturer's instructions (Roche). Briefly, after fixation in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), cryostat brain sections (20 µM) were washed three times in phosphate buffer and incubated for 30 min with a 0.1% sodium citrate, 0.1% Triton X-100 solution to increase tissue permeability. Slides were again washed, and incubated with TUNEL solution for 90 min at 37°C in a humid chamber in the dark. After washing, the slides were incubated with an anti-GFAP antibody (1:2000), in TBS containing 3% BSA and 1% Triton X-100 and left for 48 h at 4°C. The slides were incubated with Alexa Fluor anti-fluorescein-488 and -633-conjugated goat anti-mouse IgG (1:2000; Molecular Probes, Eugene, OR) in blocking buffer, both at a dilution of 1:1000. Finally, after washing, the slides were mounted in Clear Mount (Electronic Microscopy Sciences, Hartfield, PA). Immunofluorescence was visualized directly by using a DM IRB confocal microscope (Leica, Wetzlar, Germany).