Adult Mouse Digit Amputation and Regeneration: A Simple Model to Investigate Mammalian Blastema Formation and Intramembranous Ossification

Proximal digit tip amputation initiates simultaneous blastema and transient fibrosis formation and results in partial regeneration

“…For all surgical procedures, anesthetization of mice was performed using isoflurane, beginning with an initial dose of 3% isoflurane and maintained at 2% over the course of surgery. Distal terminal phalanx (P3) amputation (Figure 1A) of digits 2 and 4 from each hind limb was performed as previously described 7,31 . Proximal P3 amputation of digits 2 and 4 from each hind limb was performed using a sterile #10 scalpel to sever the digit at the amputation plane shown in Figure 1A.…”

“…Fixed digits were decalcified using Decalcifier I (Surgipath, Leica Biosystems, Richmond, Illinois) for 24 hours at room temperature. Digits were processed through a graded ethanol series, Xylenes, and paraffin as described 31 . Digits were embedded in paraffin wax and serial sectioned at 4.5 μm thickness.…”

“…Digits were embedded in paraffin wax and serial sectioned at 4.5 μm thickness. Prior to staining, slides were incubated at 65°C for 45 minutes, followed by incubation at 37°C for ≥15 minutes, and deparaffinized as described 31 . Mallory trichrome staining was performed to visualize general histology.…”

“…Slides were treated with Protein Block Solution (DAKO) for 1 hour at room temperature, followed by incubation with primary antibody at 4°C overnight. Immunostaining was performed using the following primary antibodies: rabbit anti‐ColI (Novus Biologicals, Centennial CO; NB600‐408, 1 μg/mL); mouse anti‐smooth muscle actin (Dakocytomation; M0851, 2 μg/mL); rabbit anti‐S100A4, FSP‐1 (Abcam, Cambridge, UK; ab27957, 1 μg/mL); rat anti‐mouse F4/80 (eBioscience; 14‐4801, 5 μg/mL); rabbit anti‐COL3 (Abcam; ab7778, 2 μg/mL); rat anti‐ER‐TR7 (Abd Serotec, Raleigh, NC; MCA2402, 5 μg/mL); rabbit anti‐CTSK (Abcam; ab19027, 2 μg/mL); rabbit anti‐Runx2 (Sigma‐Aldrich Co.; HPA022040, 4 μg/mL); monoclonal mouse anti‐Proliferating Cell Nuclear Antigen (PCNA) (Abcam; ab29, 0.5 μg/mL); rabbit anti‐OSX, SP7 monoclonal antibody (Abcam; ab22552, 0.125 μg/mL); rat anti‐CXCR4 (R&D Systems; MAB21651, 2 μg/mL); and rabbit anti‐human vWF8 (DAKO; A0082, 1 μg/mL), and incubated with secondary antibodies at room temperature for 45 minutes as described 31 . Digit imaging was carried out using the Olympus BX61 fluorescence deconvolution microscope, and processing of images was performed using Slidebook software (Intelligent Imaging Innovations Inc., Denver, Colorado).…”

“…Anesthetization of mice was performed as described above. Scans were carried out using the vivaCT 40 (SCANCO Medical, Wayne, Pennsylvania), at a 10.5 μm voxel size and 45 kVp energy, with 1000 projections per 180° captured at 380 milliseconds using continuous rotation, as described 7,31 . μCT‐generated images were converted to dicom files and imported to ImageJ.…”