The harvested samples were fixed immediately in 10% neutral-buffered formalin (24 hours), dehydrated using an alcohol gradient, cleared, and embedded in paraffin blocks, as reported previously (46). Histological sections (5 μm in thickness) were prepared using a microtome. H&E staining and Masson’s trichrome staining were performed according to standard procedures to examine the general appearance of soft tissues. The protein expression in the newly formed tissues was examined using immunohistochemistry. The sections were deparaffinized with the Bond Dewax Solution, rehydrated with ethanol gradient, digested with proteinase K to retrieve the antigen, and blocked with goat serum in sequence, according to the standard technical procedures of Institute of Molecular and Cell Biology Advanced Molecular Pathology Laboratory (Singapore). The sections were then incubated with the primary antibodies of monoclonal mouse antihuman COL-I (1:100 dilution in PBS; 15 min) and related IgG1 isotype (1:100 dilution in PBS; negative control, 15 min) and, subsequently, with the secondary polymers (5 min). The slides were finally counterstained with hematoxylin (7 min). The native tendon tissue samples of micropigs were also examined as positive controls for comparison. Samples were imaged using an optical microscope with the identical parameters for each staining.

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.