Avidin biotinylated enzyme complex abc

Gastrocnemius muscle and femoral arteries from VitD₃ or vehicle‐treated rats were fixed with 10% neutral buffered formalin (10% NBF). The tissues were embedded in paraffin, sectioned, and deparaffinized. Von kossa staining was used to assess calcification. H&E staining was used to evaluate the muscle fiber morphology. Verhoeff–van Gieson (VVG) staining was used to examine the integrity of elastic fibers in arteries.
To determine capillary density, CD31 immunohistochemistry staining was performed. After deparaffinized, gastrocnemius muscle sections were treated with 10 mM HIER citrate buffer (pH 6.0) for antigen retrieval and then blocked with Dako serum‐free blocking solution (Dako) followed by incubation of anti‐CD31 antibody (Santa Cruz, sc‐1506) and biotinylated anti‐goat secondary antibody. Avidin‐biotinylated enzyme complex (ABC) (Vector Laboratories) and a diaminobenzidine (DAB) substrate chromogen system (Dako) were used to detect CD31. The sections were counterstained with hematoxylin. Slides were viewed with an Olympus microscope with a digital camera. Capillaries and muscle fibers were counted manually by a blinded observer using ImageJ software in 10 different randomized fields per slide (40×) and shown as densities per high power field.

Skin was embedded fresh in Tissue Freezing Media (TFM; Triangle Biomedical), flash frozen in liquid nitrogen and immediately stored at −80 °C. Remaining treated dorsal skin was flash frozen and stored at −80 °C for use in RNA experiments. Immunohistochemistry was performed on 8 μm sections. Antibodies and isotype controls were detected using either rabbit anti-rat IgG biotinylated or goat anti-rabbit IgG biotinylated, amplified with Avidin/Biotinylated Enzyme Complex (ABC, Vector) and were visualized using the enzyme substrate diaminobenzidine (Vector). Slides were counterstained with hematoxylin and imaged as previously described^{42 (link)}. Three different observers have quantified each image manually on two occasions.