Envision system hrp polymer kit

The hepatic tissue was surgically removed from the zebrafish, fixed in 10% formalin buffer solution, and subsequently frozen. The fixed hepatic tissue was embedded in the paraffin, and 7 μm thick slices were prepared and stained with hematoxylin and eosin (H&E) [34 ].
Oil red O staining was performed as the previously described method [31 (link)]. In brief, 7 μm thick hepatic tissue sections were prepared and mounted on the slide. The tissue section was stained with oil red O solution for 15 min at 60 °C, followed by washing with 60% isopropanol. The section was stained with hematoxylin for 30 s, washed with water, and visualized under a microscope.
Immunohistochemical (IHC) staining was performed to detect proinflammatory cytokine IL-6 as the previously described method [35 (link)]. In brief, IHC primary antibody (ab9324, Abcam, London, UK) was diluted (1:200) according to the manufacturer’s instructions and incubated overnight at 4 °C with tissue section. The IHC reaction was visualized using the EnVision + System-HRP polymer kit as a secondary antibody (1:1000, Code K4001, Dako, Glostrup, Denmark). The tissue was visualized under an optical microscope (Nikon, Tokyo, Japan). Hepatic DHE staining for the ROS analysis was performed using the same protocol described in Section 2.4.

The hepatic tissue from zebrafish was excised surgically and processed, as mentioned in Section 2.6. The hepatic morphological changes and ROS production were examined by H&E and DHE staining, respectively, as mentioned in Section 2.6.
The pro-inflammatory IL-6 level in the hepatic tissue was determined by immunohistochemical (IHC) staining as previously described method [36 (link)]. In brief, the tissue section was flooded with 200× diluted IHC primary antibody (ab9324, Abcam, London, UK), followed by overnight incubation at 4 °C. The EnVision+ System-HRP polymer kit containing secondary antibody (1:1000, Code K4001, Dako, Denmark) developed the IHC stained area that was visualized under an ocular microscope (Nikon, Tokyo, Japan).

The hepatic tissue was removed from the zebrafish, fixed in a 10% formalin buffer solution, and frozen. Fixed liver tissue was processed routinely for paraffin embedding, and a 7 μm thick section was prepared and stained with Hematoxylin and Eosin (H&E) [25 (link)], as well as immunohistochemistry (IHC) [26 (link)], and observed under an optical microscope (Nikon, Tokyo, Japan), with a magnification of ×400. In the IHC staining for IL-6, the primary antibody was diluted according to the manufacturer’s instructions and incubated overnight at 4 °C (1:200; ab9324, Abcam, London, UK). The IHC reaction was visualized using EnVision + System-HRP polymer kit as a secondary antibody (1:1000, Code K4001, Dako, Denmark).