Green fluorescently labeled anti rabbit secondary antibody

Human osteoarthritis articular cartilage samples were collected from discarded tissues of total knee replacement surgery in accordance with the approval from the Institutional Review Board (IRB) of Rhode Island Hospital. Human OA cartilage tissues were fixed, decalcified, dehydrated, paraffin embedded, cut, and hydrated. The cut sections were treated by 3% hydrogen peroxide in methanol for 30 minutes, and then, digested by 100 mg/mL hyaluronidase for 15 minutes. Nonspecific protein binding was blocked using 3% bovine serum albumin (BSA) in PBS. For immunofluorescence staining, human cartilage sections were stained with primary antibodies (mouse anti-CD166 Abcam, MA, USA, catalog number: ab233750), 1:100; mouse anti-ALK3 (Santa Cruz, TX, USA, catalog number: sc-518037), 1:100; rabbit anti-ALK5 (Invitrogen, MA, USA, catalog number: PA5–32631), 1:100; rabbit anti-pSMAD1 antibody (Cell Signaling Technology, MA, USA, # 5753S), 1:100; or rabbit anti-pS-MAD2 antibody (Cell Signaling Technology, MA, USA, # 8828S), 1:100 at 4°C overnight. Sections were stained for 30 minutes with a red fluorescently labeled anti-mouse secondary antibody and/or a green fluorescently labeled anti-rabbit secondary antibody (Invitrogen, MA, USA). The VECTASHIELD Mounting Medium with DAPI was used for cell nuclear staining and section storage.