Dako envision dual link system hrp dab kit

The paraffin sections of different organs from both PPRV-infected and control goats collected at 7, 10, 14, and 18 dpi were analyzed by immunohistochemistry for the localization of PPRV antigen. A monoclonal antibody against N protein of PPR virus (clone 38-4), received as a gift from IAEA, Vienna, Austria, was used as the primary antibody (12 (link), 31 (link)). A commercial immunoperoxidase (IP) detection system, Dako EnVision® Dual Link System-HRP (DAB+) kit (Agilent Technologies, Santa Clara, CA, USA) was used to detect the bound primary antibodies. The paraffin sections were mounted on poly-l-lysine coated slides (Sigma, St. Louis, MO, USA). The IP staining was performed as described in the kit literature. With each group of staining, sections from the uninfected control group were also stained similarly to serve as the negative control.

Ovarian immunohistochemistry was performed as described previously (Subramanian et al., 2020). Ovaries were fixed in 4% paraformaldehyde (w/v) (Sigma), embedded in paraffin and serially sectioned at 5 μm and mounted on microscope slides (ThermoFisher). Antigen retrieval with 10 mM sodium citrate (Sigma) was used prior to incubation with primary anti‐mouse Vasa homolog (MVH) antibody (1:1000; Abcam). Endogenous peroxidase activity was quenched with 3% hydrogen peroxide (Sigma) followed by incubation with secondary HRP‐conjugated antibody and detected for HRP signals according to the manufacturer's instructions in the Dako EnVision Dual Link System‐HRP (DAB+) kit (Agilent Technologies). Sections were counterstained with Gill's Haematoxylin for 10 s. Every fifth section was photographed using a slide scanner (ScanScope XT; Leica Biosystems). Follicles at four different developmental stages (primordial, primary, secondary and antral) were classified as previously described (Myers, Britt, Wreford, Ebling, & Kerr, 2004). Follicles of every fifth section were counted as described before (Subramanian et al., 2020; Tilly, 2003).