Mouse anti vimentin monoclonal antibody

During cell culture, we confirmed the identity of tenocytes using the ICC with 2 × 10⁵ cells. After transferring 200 μL of cell culture to the 8-wells of a chamber slide, the cells were grown to confluence with the addition of fresh media for one day. After washing with PBS, the cells were fixed with 4% formaldehyde. Permeabilization of the membranes was done by incubating the slides with 0.25–0.5% Triton X-100 in PBS for 10 min, followed by blocking with rabbit normal serum, donkey normal serum, for vimentin and tenomodulin, respectively. The cells were incubated in the dark with mouse anti-vimentin monoclonal antibody (1:100 dilution; Dako, Glostrup, Denmark; code: M0725) and goat anti-tenomodulin antibody (1:25 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA; code: sc-49325) for 60 min at 37 °C. After additional washing, the secondary antibodies were incubated in the dark for 30 min at 37 °C, and mounted in a Vectashield H-1000 mounting medium for fluorescence (Vector Laboratories, Burlingame, CA, USA). Analysis was done using an Olympus CKX41 microscope equipped with epifluorescence and an Olympus XC10 digital camera. To determine the expression of vimentin or tenomodulin in the cultured cells, we randomly sampled 40 views of cell staining from two groups (healthy tenocyte, tendinopathy tenocyte), with approximately 1000 cells each.

Unless stated otherwise, all chemicals were obtained from Prestwick Chemical. The antibodies used in the current study include mouse antilamin monoclonal antibody (sc-7292; Santa Cruz Biotechnologies, Heidelberg, Germany), mouse antitubulin monoclonal antibody (T5168; Sigma-Aldrich, Saint-Quentin Fallavier, France) and mouse antivimentin monoclonal antibody (M0725; Dako, Glostrup, Denmark). The far red-fluorescent lectin GS-II Alexa Fluor^® 647 conjugate was obtained from Thermofisher Scientific (Darmstadt, Germany).