Rat anti β4 integrin

Immunofluorescence labelings were performed on 5 μm frozen sections as previously described [21 (link)]. Primary antibodies and dilutions used for immunofluorescence labeling were rabbit anti-CD151 (LAI-2) at 1:500 [21 (link)]; rat anti-CD31 (BD Biosciences, Franklin Lakes, NJ, USA) at 1:200; rabbit anti-α3 integrin (a kind gift from Dr. Fiona Watt, Wellcome Trust Centre for Stem Cell Research, Cambridge, UK) at 1:1000; rat anti-β1 integrin (BD Biosciences) at 1:200; rat anti-β4 integrin (BD Biosciences) at 1:200; rat anti- α6 integrin (Chemicon, Temecula, CA, USA) at 1:200. The double labeling where two rabbit primary antibodies were used (CD151 and α3 integrin) was performed sequentially following established methods as described previously [21 (link)].

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.