Rabbit anti phh3

For immunofluorescent staining, 10 uM thick sections were fixed for 8 min in 4% PFA in PBS-T (containing 0.2% Triton X-100) or 3 min in −20C methanol. Samples were washed for 5 min in PBS-T, and then incubated in blocking buffer (5% normal goat serum, 5% normal donkey serum, and 3% bovine serum albumen in PBS-T) for 15 min. Sections were then incubated with primary antibody overnight at 4C (for anti-K5/K14), or for 15 min at room temperature (all other antibodies). After washing with PBS-T, sections were incubated with secondary antibody for 10 min at room temperature. Sections were washed again and then mounted in 90% glycerol in PBS with 2.5 mg/mL p-Phenylenediamine (Sigma-Aldrich). Antibodies used were: rat anti-β4-integrin (BD Biosciences), rabbit anti-pHH3 (Cell Signaling Technology), rat anti-BrdU and rabbit anti-NuMA (Abcam), chicken anti-Keratin5/Keratin14 (Lechler lab), rabbit anti-Keratin 10 (Covance). Images were collected using a Zeiss Axio Imager Z1 fluorescence microscope with Apotome attachment. Adobe Photoshop and ImageJ software were used to process images.

For the immunofluorescence analysis of kidney sections, 3-μm periodate-lysine paraformaldehyde (PLP)-fixed cryosections were air dried. To detect phospho-Histone H3 antibody (pHH3), antigen retrieval was performed in citrate buffer 10 mmol/L (pH 6.0) at boiling temperature for 20 min, followed by incubation with citrate buffer (20 min) at room temperature to enhance the reactivity of antibodies to antigens. Slides were washed with PBS 1× and incubated with 1% BSA to block nonspecific sites. Rabbit anti-pHH3 (1:75; #9701, Cell Signaling, Danvers, MA, USA) was used followed by the specific Cy3-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, Cambridge, UK). Nuclei were stained with 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI, 28718-90-3, Sigma-Aldrich, St. Louis, MO, USA) and the renal structure with fluorescein wheat germ agglutinin (WGA; FL-1021, Vector Laboratories, Burlingame, CA, USA). Finally, slides were mounted using Dako Fluorescence Mounting Medium (S3023, Agilent Technologies, Santa Clara, CA, USA) and examined with an inverted confocal laser scanning microscope (Leica TCS SP8, Leica Microsystems, Wetzlar, Germany). Negative controls were obtained by omitting primary antibodies on adjacent sections. pHH3-positive cells in kidney tissue were evaluated in at least 10 HPF/section (n = 3 mice for each group).