Ab756p

Slides containing planarian sections were fixed with 4% formaldehyde for 20 min. They were washed thrice in PBS and subsequently blocked for 1 h in 10% horse serum in PBSTx (PBS+0.3% Triton-X). Overnight incubation with primary antibody was performed at 4°C (rabbit anti-mouse collagen IV, Abcam, ab-756p; 1:200). Secondary antibody was incubated for 45 min (Alexa-Fluor anti-rabbit 488, Molecular Probes). Sections were then washed and stained with DAPI.

Whole-mount staining of retinas was performed as previously described (83 (link)). Briefly, eyeballs were removed and fixed in 4% PFA in PBS for 1 hour at 4°C. Eyeballs were washed 3 times in 1× PBS, and retinas were dissected, permeabilized in 1% TritonX-100/PBS (PBST) at room temperature for 30 minutes, and blocked with CAS-block (Thermo Fisher Scientific, 88120) at room temperature for 30 minutes. Primary antibodies were diluted in CAS-block and incubated at 4°C overnight, followed by incubation in appropriate secondary antibodies for 4 hours at room temperature. Retinas were washed in 3 times in 1× PBS and mounted on a slide using the ProLong Diamond Antifade Mountant (Thermo Fisher Scientific, P36961). The antibodies used were antibodies to IB4 (Thermo Fisher Scientific, I21411, 1:250), RFP (Abcam, ab62341), ERG1/2/3 (Abcam, ab196149, 1:200), GM130 (BD Biosciences, 610822, 1:100), Ki67 (Cell Signaling Technology, 9449, 1:100), COLIV (MilliporeSigma, AB756P, 1:100), FN (Abcam, Ab2413, 1:100), and cCASP-3 (Cell Signaling Technology, 9661, 1:100).

The tail skin sections were subjected to immunostaining as previously reported [12 (link)]. Primary antibodies were used at the following dilutions: rabbit anti-K14 (1:1000, BioLegend, 905304), goat anti-PDGFRα (1:100, R&D systems, AF1062), rat anti-α6-integrin (1:150, BD biosciences, 555734), rabbit anti-Collagen IV (1:100, Chemicon, AB756P), rat anti-BrdU (1:300, Abcam, ab6326), mouse anti-K10 (1:500, Abcam, ab9026) and guinea pig anti-K31 (1:100, PROGEN Biotechnik, GP-hHa1). To stain with anti-BrdU antibody, skin sections were incubated with 1N HCl at 37°C for 1 hour after blocking. Preparations were analyzed and imaged using a fluorescent microscope (Zeiss, Axio Imager.Z2). The brightness and contrast of images were adjusted with equal intensity among different mouse groups by using Adobe Photoshop.