Dako envision detection system kit

The staining of liver tissues was performed as previously described [1 (link)]. Briefly, mice livers were fixed in 10% formalin for 24 h and then embedded in paraffin. For IHC of hepatic TNFα, MMP-9, HSL/p-HSL and ATGL, antigen retrieval of deparaffinized sections was performed in Dako target retrieval solution, pH 9.0 in a vegetable steamer followed by quenching of endogenous peroxidase activity with 3% H₂O₂ in methanol. Sections were then incubated with specific primary antibodies overnight at 4 °C in a humidified chamber. The antibodies of HSL/p-HSL (Cell Signaling, Boston, MA, USA), MMP-9 (Abcam, Cambridge, UK), TNFα (Abcam) and ATGL (Cell Signaling) were used. The sections were then examined using a DAKO EnVision Detection System kit (DAKO, Carpinteria, CA, USA) and counterstained with hematoxylin. Images were obtained through a Nikon Eclipse TE2000-S microscope.
For the staining of macrophages infiltrated into liver tissues, F4/80 and CD11c (Abcam) were used to stain M1 macrophages, and CD206 and CD209a (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used to stain M2 type macrophages.

Tissue microarray blocks were cut into 4 µm thick slices, and the sections were used for immunohistochemical staining. Briefly, TMA slices were deparaffinized with xylene, hydrated in a graded ethanol series, and then treated with peroxidase-blocking solution (Dako, Copenhagen, Denmark) for 10 min to block endogenous peroxidase, as previously described [21 (link)]. The sections were blocked with protein block solution (Dako) for 60 min at room temperature (RT) and then incubated with rabbit polyclonal antibody-PrP (8H4; Sigma-Aldrich, St. Louis, MO, USA) diluted antibody diluent. After primary antibody incubation, the sections were detected using the Dako EnVision Detection System kit (Dako) according to the manufacturer’s instructions.