Click it edu

For fluorescence imaging, immunofluorescence, and in situ hybridization of sections, hindlimbs were fixed in 4% paraformaldehyde and frozen in OCT medium. Alternating transverse cryosections (12 μm) were collected across the length of the limb to capture the trajectory of the Achilles tendon from skeletal insertion to muscle. Immunostaining was carried out using antibodies against laminin (Sigma) and α-smooth muscle actin (Sigma) with Cy3 or Cy5 secondary detection (Jackson ImmunoResearch), with DAPI counterstaining to visualize cell nuclei. EdU and Tunel assays were performed using the Click it EdU (Life Technologies) and In Situ Cell Death Detection kits (Roche), respectively, according to manufacturer’s instructions.
For analysis of longitudinal sections, hindlimbs were fixed in zinc formalin, dehydrated, and infiltrated with methacrylate monomer and embedded. Plastic sections were then acquired at 6 μm, stained with Picrosirius Red, and imaged with polarized light to visualize collagen alignment. Additional sections were immunostained for type I and III collagens (Abcam) with DAB Chromagen secondary detection (Vector Laboratories) and counterstained with Toluidine Blue.
All images were acquired using Zeiss Axio Imager microscope; an Apotome was used for optical sectioning of fluorescent images.

Mice were trans-cardially perfused and fixed using 1X PBS⁽⁻⁾ and 4% paraformaldehyde (PFA), respectively. Brains were cryoprotected in 30% sucrose prior to OCT embedding. For co-localization staining, 12 μm frozen sections were processed for immunostaining with antibodies against H3K9me3 (Upstate #07-422; 1:500), Glial Fibrillary Acidic Protein (GFAP)—clone GA5 (Millipore #MAB3402; 1:500), Vimentin (Sigma V2258; 1:500), Polysialic Acid-NCAM (PSANCAM)—clone 2-2b (Millipore #MAB5324; 1:500), Mash1(Abcam #ab38556; 1:500), and EdU 5-ethynyl-2′-deoxyuridine (Life Technology, #C10337 Click-iT® EdU).