Dmlb microscope

Liver samples were fixed in 4% buffered formalin and embedded in paraffin. Deparaffinized tissue sections were stained with H&E using standard procedures and images were obtained using a high-resolution Leica DC200 digital camera mounted on an Olympus DMLB microscope. Immunohistochemistry of the macrophage marker F4/80, a kind gift of Dr. Antonio Castrillo, was performed on 4 μm deparaffinized-rehydrated sections with 1:100 antibody dilution. Antigen retrieval was carried out with citrate buffer and endogenous peroxidase activity was inhibited with 3% H₂O₂. Samples were blocked and incubated overnight with the antibody, and signal was amplified with the ABC Kit (Vectastin). Slides were revealed with DAB (Vector), counterstained with Hematoxylin and mounted with DePeX (Serva).

After euthanization, the Achilles tendon of each rat was dissected carefully from the attached calcaneal bone. The samples were fixed using 4% buffered formalin, embedded in paraffin, and stained with H and E and Alcian blue to quantify the severity of tendinopathy. Tissue changes were evaluated by histological criteria according to the Movin score. Images were obtained using a high-resolution Leica DC200 digital imaging system (Leica Microsystems, Wetzlar, Germany) mounted on an Olympus DMLB microscope. The Movin score was used to quantify the amount of tendon degeneration using H and E and Alcian blue staining (Supplementary Table S1) [36 (link),37 (link)]. Immunohistochemistry with SP antibody (ab14184, Abcam, Cambridge, MA, USA) was performed on 5-μm-thick formalin-fixed, paraffin-embedded blocks on a Bond-III Automated IHC Stainer (Leica Biosystems). The SP antibody was diluted (1:1000) and the BOND Polymer Refine Detection Kit was used according to the manufacturer’s instructions. ImageJ software (National Institute of Health, Bethesda, MD, USA) was used to quantify the amount of SP expression after immunohistochemical staining.