Fluormount g

Eyes from P5 mice were collected and fixed in 4% paraformaldehyde (3–10 minutes) and stored at −20 °C in methanol until use. Next, the dissected retinas were washed in 1xPBS for 30 minutes then followed by a 30 minutes in 3% paraformaldehyde and then washed three times in 1xPBS. To assess the spreading of the superficial layer retinas were wholemount stained with anti-collagen IV and mounted with Fluormount-G (Southern Biotech, Birmingham, AL, #0100-01). For quantification, the distance the retinal vessels had spread from the optic nerve head to the periphery of the retina relative to the entire retinal radius was determined for each quadrant of the retina at P5 which was then recorded as an average for each retina. Tip cell sprouts were determined as we previously described^{31 (link)}. The distance the superficial layer had spread was quantified from digital images taken on an EVOS microscope as we previously described^{12 (link)}.

For immunocytochemistry, cells were cultured under different conditions for 48 h and fixed in 2% paraformaldehyde for 30 min. Cells were treated with 0.1 M glycine for 1 min and then blocked and permeabilized with 10% FBS solution containing 0.01% saponin for at least 2 h. Thereafter, cells were incubated with primary antibodies or biotinylated HA binding protein (HABP, Millipore, USA). To evaluate the number of LESCs within the cultures, antibodies for putative stem cell markers K19 (ab166857; Abcam, Cambridge, MA, USA), K15 (ab185627; Abcam, Cambridge, MA, USA) and ΔNp63 (ab166857; Abcam, Cambridge, MA, USA) were used. In order to analyze CD44 expression, anti-CD44 (5D2-27, DSHB) was used. The cells were then washed and incubated with relevant secondary antibodies conjugated with Alexa®488 or Alexa®555, or with NeutrAvidin®555 in the case of biotinylated HABP. Thereafter, nuclei were labeled with DAPI, cytoskeleton labeled with Phalloidin®Alexa Fluor 647 (ThermoFisher Scientific, Eugene, OR) and coverslips mounted with Fluormount-G® (Southern Biotechnology Associates, USA). The immunolabeled cells were scanned under an LSM 800 confocal microscope using the Zen Image software (Zeiss) and quantified using ImageJ software (National Institutes of Health, Bethesda, MD).

Paraffin embedded tissue samples of normal lung biopsies were obtained from the Department of Pathology, Landspitali University Hospital. The samples were deparaffinized, refixed in methanol/acetone (1/1) for 5 minutes at −20°C and stained with EnVision®+ System-HRP kit (Dako). Primary antibodies (ncl-p63 (clone 7JUL) from Leica Microsystems and ΔNp63, provided by Dr. Sat Sinha, (University of Buffalo, NY) were incubated overnight at 4°C. Cell cultures were fixed in 3,7% formaldehyde. For nuclear protein staining, cells were also fixed in methanol/acetone (1/1) for 5 minutes at −20°C. They were then washed two times for 10 minutes at room temperature with PBS, and blocked with 10% goat serum in IF-buffer (0.2% Triton X-100; 0.1% BSA and 0.05% Tween-20 in PBS). Primary antibodies (ncl-p63 (clone 7JUL)) and ΔNp63, (RR-14, [7] (link)), were incubated overnight at 4°C, followed by three 10 minute washes in PBS. Isotype specific secondary antibody conjugates Alexa Fluor® (Invitrogen) were incubated for 2 hours at RT. After PBS rinsing, nuclear staining was performed with TO-PRO-3® (Invitrogen) for 30 minutes followed by 3×10 minute washes. The samples were then embedded in Fluormount-G (Southern Biotech, Birmingham, AL) for microscopic analysis.