Publication protocol
Following dissection, knees were fixed in 4% paraformaldehyde, decalcified using a 14% EDTA solution, processed for paraffin embedding in a frontal orientation, and cut into 7-μm alternate sections. The ImmunoCruz ABC Staining System (Santa Cruz Biotechnology, Dallas, TX) was used for all immunohistochemical staining. After sections were deparaffinized and rehydrated, antigen retrieval was done by incubating sections with 10mM sodium citrate buffer for 10 minutes at 95°C. To reduce endogenous target, slides were blocked with 3% hydrogen peroxide in water for 15 minutes, then blocked for 1 hour with 10% serum, and incubated with primary antibodies in blocking buffer at 4°C overnight. The following primary antibodies were used: goat anti-sclerostin (SOST) (1:100; R&D Systems, Minneapolis, MN; AF1589; RRID: AB_2195345), rabbit anti-pLRP6 (1:100; Bioss, Woburn, MA; bs-3253R; RRID: AB_10858068), rabbit anti-pLRP5 (1:100; Abcam, Cambridge, MA; ab203306; RRID: AB_2721918), goat anti-WNT5A (1:50; R&D Systems; AF645: RRID: AB_2288488), rabbit anti-WNT7B (1:100; GeneTex, Irvine, CA; GTX114881; RRID: AB_11172941), rabbit anti-pGSK3β (1:50; Abcam; ab75745; RRID: AB_1310290), rabbit anti–lymphoid enhancer binding factor 1 (LEF1; 1:50; Sigma-Aldrich, St. Louis, MO; AV32404; RRID: AB_1852782), rabbit anti–non-phospho β-catenin (1:50; Cell Signaling Technology Inc., Danvers, MA; 8814; RRID: AB_11127203), rabbit anti-MMP13 (1:100; Abcam; ab39012; RRID: AB_776416), and goat anti-MMP19 (1:100; Santa Cruz Biotechnology; sc6840; RRID: AB_2144734). A negative control slide was used each time, which was incubated overnight with only blocking serum. Sections were then washed and incubated with the appropriate 1:200 biotinylated secondary antibody (Vector Laboratories, Burlingame, CA) for 30 minutes. Finally, slides were washed, developed with 3,3′-diaminobenzidine (DAB) Peroxidase Substrate Kit (Vector Laboratories), and counterstained with Harris hematoxylin (Thermo Fisher Scientific, Waltham, MA). Quantitative analysis of DAB-positive vs total cells was performed using a web-based automated image analysis application (ImmunoRatio). Images were uploaded and analyzed as recommended by ImmunoRatio application from three images per sample, as described (21).
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