Immu mount mounting medium

Non-radioactive in situ hybridization of fixed sections from dorsal skins was performed with Dig-labeled RNA probes corresponding to nucleotides (nts) 592-931 of Ccnd1 (GenBank Acc. No. NM_007631), nts 465-943 of Fgf7 (NM_008008), nts 699-1211 of Fgf10 (NM_008002), nts 717-1213 of Rspo1 (NM_13868), nts 754-1169 of Rspo2 (NM_001357956), nts 921-1348 of Rspo3 (NM_028351), and nts 300-760 of Rspo4 (NM_001040689).
To generate the probes, plasmids containing the corresponding templates were linearized and used for in vitro transcription with T7 RNA polymerase (Roche cat#10881767001) in the presence of Dig-labeled UTP (DIG RNA Labeling Mix, Roche cat#11277073910). BMpurple substrate (Roche, cat#11442074001) was used for signal detection and Immu-Mount mounting medium (Thermo Fisher Scientific cat#9990402) was used for mounting.

Submandibular glands and SGm were isolated and fixed in 4% paraformaldehyde overnight at 4 °C. Tissues were paraffin-embedded and then cut into 5 μm sections. Slides were treated with HIER buffer (10 mM sodium citrate, 0.05% Tween-20, pH 6.0) for antigen retrieval in a pressure cooker for 10 minutes then sections were blocked in CAS-block histochemical reagent (Thermo Fisher Scientific, 008120). Permeabilization was performed with 0.5% Triton X-100 in PBS for 5 minutes. Immunostaining was performed overnight (at 4 °C) with primary antibody for γH2AX (EMD Millipore, 05–636). Alexa-Fluor 594-conjugated donkey anti-mouse IgG was diluted 1:500 (Invitrogen, A21203) as secondary antibody and applied on sections for 1 hour at room temperature. Following a PBS rinse, 10 μg/ml DAPI (Invitrogen, Carlsbad, CA) in PBS was applied to sections for 5 minutes. Sections were washed thrice in PBS for 5 minutes and the slides were mounted using Immu-Mount mounting medium (Thermo). Microscopic images were acquired using a Leica TCS SP5 confocal microscope with a 100X oil immersion objective and Argon laser. Analysis of images was performed in ImageJ.

For X-gal staining, dorsal skins were harvested, stretched on a membrane to keep the skin flat, embedded in OCT compound, immediately fresh frozen on liquid nitrogen and cryosectioned (20 µm). Sections were fixed for 10 min in 0.2% glutaraldehyde (Sigma Aldrich cat#G7776), washed three times for 5 min in PBS, and incubated with 1 mg/ml X-gal (Sigma Aldrich, cat#B4252) overnight at 37 °C. Sections were washed three times for 5 min in PBS and mounted with Immu-Mount mounting medium (Thermo scientific).

For Figs. 2, 3 and 6g, fixed brain sections were washed with PBS three times at room temperature (20–25 °C) prior to the procedure to remove the sucrose and freezing compound residue. The sections were then incubated with blocking solution (5% goat serum, 0.5% (v/v) Triton X-100, PBS) for 1 h at room temperature, then with the primary antibody and 4,6-diamidino-2-phenylindole (DAPI) diluted in blocking buffer for 16–20 h at 4 °C, washed in PBS three times for 30 min and incubated with secondary antibody diluted in the blocking buffer for up to 4 h at room temperature. Next, sections were incubated with PBS for 30 min three times and mounted with a cover glass (Menzel-Gläser) and Immu-Mount mounting medium (Shandon, Thermo Scientific). For experiments with dual mRNA and protein labeling, instead of mounting after the hybridization protocol, the sections were subjected to the IHC as described here. For IHC staining in Fig. 6e,f, the previously described procedure was used^{18 (link)}.

Five separate wells with cover slips were prepared for each oxygen condition (Norm, IH and SH, total 15 wells) at the re-plating step on day 3, and from day 3 to 7 an individual well in each condition was pulsed with bromodeoxyuridine (BrdU) (Sigma-Aldrich) at a 100 mmol concentration for 6 h, thereby providing a “snapshot” of the frequency of proliferation occurring for a 6 h period on each day in culture (10 (link)). After the last cycle on each day the supernatant was removed and colonies were fixed with ice-cold methanol for 10 min, washed with PBS and immersed in 2 M HCl solution for 2 h. Then the cells were washed 3 times with 0.1 M boric buffer and PBS before overnight incubation with anti-BrdU antibody (1:10) (Roche, Germany). Afterwards, the cells were stained with AEC staining mix kit (Sigma-Aldrich), washed with PBS and additionally stained with hemotoxylin, following by mounting with Immu-Mount mounting medium (Thermo, Pittsburg, PA, USA). Slides were analyzed with Olympus CX4 microscope at x10 magnification. Proliferation was expressed as the proportion of BrdU positive cells to EC-CFUs volume sum per well (cells/μm³). Colony volume (V) was calculated using the formula V = (πr²h)/3, where r = the radius of the base of the colony and h = the height of the colony, assumed to be equal to r (10 (link)).