Brdu solution

Fish were anesthetized, and the distal portion of the caudal fins were amputated proximal to the first lepidotrichia branching point. Following amputation, fish were revived and returned to the housing system. All fin amputations were perpendicular to the anterior/posterior plane to avoid uneven fin outgrowth from the dorsal or ventral halves of the fin. Experimental fins were imaged prior to amputation, and at 4, 6, 12, 19, and 32 days post amputation (dpa). For the proliferation assay, fish were immersed in a 5 mM BrdU solution (Sigma Aldrich, St. Louis, MO) from 3 to 4 dpa as previously described [40 (link)].

For the in vivo BrdU incorporation assay, 100 μL of 10 mg/mL BrdU solution (Sigma) was injected intraperitoneally. Mice were sacrificed 4 h after the injection, and colon tissues were harvested.
For immunohistochemical staining of BrdU, Ki-67, Bmi1, TERT, and IGF-1 in samples of acute DSS colitis model, tissues were fixed by 4% paraformaldehyde and embedded in paraffin. Tissues were then sliced into 4 µm-thick sections, and antigens were retrieved using citrate solution. Anti-Ki-67, -IGF-1, -Bmi1, -TERT (Abcam), and -BrdU (CST) were used as primary antibodies and incubated at 4 ºC overnight. Goat-anti-rabbit IgG was used as a secondary antibody and diaminobenzidine (DAB) staining was performed. Sections were scanned using the Aperio ScanScope CS System and positive rates were calculated by Image J software (National Institutes of Health).

Brain sections and staining experiments were performed as in19 (link) except those performed on Gsh2-Cre; RCE; Zeb2^fl/fl mice brains processed as in48 (link). Primary antibodies used are: Calretinin (rabbit, Swant, 1/1000), GFP (chicken, Aves, 1/500), mouse IgG1 anti-NeuN (Millipore, 1:100), rat Igg2a anti-BrdU (AbD Serotec (Oxford B), 1/1000). Images were taken using a fluorescence microscope (Axiolmager Z1, ApoTome system, Zeiss) except for Gsh2-Cre; RCE; Zeb2^fl/fl sections (Leica DMR microscope) and for spine density measurement (laser confocal scanning microscope, LSM510, Zeiss - magnification: 63x). Data in graphics are presented as mean ± s.e.m of values obtained on n samples (*P < 0,05. **P < 0,01, ***P < 0,001). For BrdU incorporation analysis, animals at 2 dpe were injected once with a BrdU solution (50 μg/g body weight, Sigma, Saint-Louis MO) 2 hours before perfusion. BrdU staining was performed after 15 min incubation at 37° in 2N HCl-0.5%. In Fig. 4c, Zeb2 expression level per transfected cell was assessed as follows. Transfected cells in the RMS were identified based on GFP expression. Quantification of Zeb2 staining was performed using ImageJ software on a single z-plan focused on the nucleus (chosen using DAPI staining). A ROI was subsequently drawn inside the nucleus area and the mean intensity of Zeb2 staining signal was then measured across the ROI.

Fertilized White Leghorn chicken eggs (Ozark Hatchery, Meosho, MO, USA) were placed in a rocking incubator at 37°C (Kuhl, Flemington, NJ, USA) until appropriate age of development. After incubation embryos were staged according to the criteria of Hamburger and Hamilton (1951) (link), and embryos were selected that were healthy and developing normally. The adult human metastatic cutaneous melanoma cell line C8161 and its poorly aggressive isogenic counterpart, C81-61, were isolated from an abdominal wall metastasis (Welch et al., 1991 (link)) and maintained as previously described (Hendrix et al., 2002 (link)). All cultures were determined to be free of mycoplasma contamination using a polymerase chain reaction-based detection system (Roche, Indianapolis, IN, USA). For certain experiments, tumor cell drops were generated by trypsinizing the cells (as for passaging) and placing 25 μl drops of resuspended c8161 cells on the inside surface of a 60×15 mm petri dish. 3 ml of media was placed in the bottom of the petri dish and the lid was then replaced, creating hanging drops of melanoma cells which were incubated for 24-48 h in a 37°C incubator supplied with 5% CO₂. For cell proliferation experiments, 10 μl of 1 mM BrdU solution (Sigma-Aldrich, St. Louis, MO, USA) was added to cells in culture for 30 min, followed by fixation in 4°C and processed as below.

Fetal mice were injected with BrdU solution (Sigma, USA, 20 mg/ml, dissolved in 0.1 M PBS) intraperitoneally (200 mg/kg body mass). Two hours later, the E15.5 to E18.5 fetal mice were removed. Cell proliferation was determined by counting the number of BrdU-positive cells per 100 μm in the epidermis after BrdU immunohistochemistry. Cell apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) using an apoptosis cell detection kit (Trevigen, USA). The number of TUNEL-positive cells was recorded using the above approach. Detailed methods for these assays have been described previously 22 (link).