Ultraview red detection kit

Formalin-fixed, paraffin-embedded tissues were sectioned at 5-um thickness and stained with hematoxylin and eosin or by IHC as previously described [43 (link)]. Antigen retrieval was achieved by autoclaving in citrate buffer solution (DAKO Target Antigen Retrieval) and IHC for PrP^CWD was performed on an automated immunostainer (Ventana Medical Systems) using the monoclonal antibody F99/97.6.1 (VMRD) and Ultraview Red detection kit (Ventana Medical Systems). Positive and negative staining were differentiated based on comparisons with positive and negative deer control tissues included in each run, and negative control tissues showed no immunoreactivity. The extent of PrP^CWD deposition in obex sections was graded (- to ++++) using previously published criteria [31 (link)].

A representative tissue block, when available, was selected for immunohistochemistry, including double Ki67/melan A staining (Ki67: DAKO, Glostrup, Denmark, clone MIB-1, dilution 1:50; melan A: DAKO, Glostrup, Denmark, clone A103, dilution 1:10) and HMB-45 (DAKO, Glostrup, Denmark, clone HMB-45, dilution 1:20). Deparaffinization, antigen retrieval, and staining with antibodies were performed in an automatic immunostainer (Benchmark XT, Ventana Medical Systems Inc., Tucson, USA) in combination with treating sections with secondary antibodies and color development with horseradish peroxidase (ultraVIEW DAB Detection Kit, Ventana Medical Systems Inc., Tucson, USA) for Ki67 and alkaline phosphatase (ultraVIEW Red Detection Kit, Ventana Medical Systems Inc., Tucson, USA) for HMB-45 and melan A.