Goat anti prox1

Mice were anaesthetized with an overdose of sodium pentobarbital and transcardially perfused with saline, followed by 4% paraformaldehyde (PFA). Brains were post-fixed for 2 h at 4°C. Brains were sectioned on a sliding microtome or a vibratome at 60 μm. All primary and secondary antibodies were diluted in PBS containing 0.25% Triton X-100 and 2% BSA. The following antibodies were used rabbit anti-Calretinin (1:1000, Swant), chicken anti-GFP (1:3000, Aves Lab), goat anti-Prox1 (1:300, R&D systems), mouse anti-reelin (CR-50, 1:300, MBL international) and rat anti-RFP (1:500, ChromoTek). We used Alexa Fluor-conjugated secondary antibodies (Jackson ImmunoResearch Labs and Molecular Probes).

Back skin samples were embedded in OCT (Leica Biosystems, Newcastle, UK) and frozen in liquid nitrogen. Further, 10-μm frozen sections were fixed in 4% paraformaldehyde for 15 min at room temperature (RT), washed in PBS and incubated with blocking solution (5% donkey serum, 0.2% BSA and 0.3% Triton X-100 in PBS) for 1 h at RT. Next, the sections were stained with goat anti-Prox1 (R&D systems, Minneapolis, MN, USA) and rabbit anti-Sostdc1 (Abcam, Cambridge, MA, USA) antibodies overnight at 4 °C. After several washes, the sections were then incubated with Alexa Fluor 488 donkey anti-goat (Thermo Fisher Scientific, San Jose, CA, USA) and Alexa Fluor 594 donkey anti-rabbit (Thermo Fisher Scientific) antibodies for 30 min at RT. Hoechst 33342 (Invitrogen, Carlsbad, CA, USA) was used for nuclear staining. Immunofluorescence images were acquired with a Zeiss LSM 710 FCS confocal microscope (Carl Zeiss, Jena, Germany).

Back skin samples were embedded in OCT (Leica Biosystems, Newcastle, UK) and frozen in liquid nitrogen. 10-μm frozen sections were fixed in 4% paraformaldehyde (PFA) for 15 min at room temperature (RT), washed in PBS and incubated with blocking solution (5% donkey serum, 0.2% BSA and 0.3% Triton X-100 in PBS) for 1 h at RT. Next, the sections were stained with primary antibodies overnight at 4°C and, after several washes, incubated with secondary antibodies for 30 min at RT. Primary antibodies were as follows: rat anti-CD31 (BD Biosciences, San Jose, CA, USA), rabbit anti-LYVE-1 (Angiobio, Del Mar, CA, USA), goat anti-LYVE-1 (R&D Systems), rat anti-CD68 (Abcam, Cambridge, MA, USA), goat anti-podoplanin (R&D Systems), rabbit anti-cytokeratin 15 (Abcam), rat anti-Foxp3 (eBioscience, San Diego, CA, USA), and goat anti-Prox1 (R&D systems). Secondary antibodies (all from Thermo Fisher, San Jose, CA, USA) were as follows: donkey anti-rabbit Alexa Fluor 488, 594 or 647, donkey anti-rat Alexa Fluor 488 or 594, donkey anti-goat Alexa Fluor 488 or 594, and chicken anti-goat Alexa Fluor 647. Hoechst 33342 (Invitrogen, Carlsbad, CA, USA) was used for nuclear staining. Immunofluorescence images were acquired by an Axioskop 2 mot plus microscope (Carl Zeiss, Jena, Germany) and Z stacks of images were obtained using a Zeiss LSM 710 FCS confocal microscope.