Dako envision flex target retrieval solution high ph

IHC was performed on bovine foot skin from cows without BDD lesions (lesion stage M0, n = 3), acute BDD lesions (lesion stage M2, n = 3) and chronic BDD lesions (lesion stage M4, n = 3) (Döpfer et al., 1997 (link)) to detect expression of CFB, RND1, IL-6 and IL-8 in vivo. Sections (4 µm) from archived paraffin embedded tissue blocks were mounted onto glass slides, deparaffinized and antigen retrieval carried out using DAKO EnVision™ FLEX target retrieval solution high pH (Agilent) at 95°C for 25 min. Using DAKO Autostainer Link 48 (Agilent), sections were washed, and endogenous peroxidase blocked, prior to incubation (20 min) with corresponding primary antibodies at optimal dilutions for bovine skin (Table 1). IHC antibodies were each tested and their labeling optimized on appropriate tissues before use. Both negative antibody (no primary antibody) and internal labeling controls were used to confirm positive labeling and determine background staining. Sections were incubated (20 min) with label polymer, DAKO EnVision™ FLEX/HRP and bound antigen subsequently detected using DAKO EnVision™ FLEX DAB + chromogen. Sections were counterstained with hematoxylin. IHC using an anti-DD treponeme antibody (Table 1) was also performed, as previously described (Crosby-Durrani et al., 2016 (link)), to confirm the presence of DD treponemes in the sections.