Ab204178

Immunohistochemistry (IHC) experiments were performed as described [27 (link)]. Briefly, tumor sections were incubated with RCN3 antibody (Abcam Cat# ab204178—1:500 dilution), Ki67 antibody (Abcam Cat# ab16667—1:100 dilution), or CD44 antibody (Cell Signaling Cat# 37259S—1:200 dilution) overnight, followed by secondary antibody incubation for 30 min (Vector Labs Cat# MP-7500). Immunoreactivity was detected using DAB staining, and sections were counterstained with hematoxylin.

For immunohistochemisty, mouse hindlimbs were collected during postnatal tendon maturation from P10 to P60 and fixed in 10% neutral buffered formalin overnight at 4 °C. Samples were paraffin-embedded and sectioned at 6 μm, following decalcification in 10% (w/v) EDTA (pH 7.4) for 2 weeks with daily solution changes. The paraffin sections of the patellar and Achilles tendon incubated with antibodies against Rcn3 (Abcam, ab204178) at 4 °C overnight. The sections were then incubated in anti-rabbit-biotin antibody (Jackson Immuno, 711-065-152) for 30 min at RT, rinsed with PBS, and incubated with streptavidin-HRP (Jackson Immuno, 016-030-084) for 30 min at RT. Bounded antibodies were detected using NovaRed substrate (Vector Laboratories, SK-4800).