Epitope retrieval solution and steamer

Paraffin-embedded liver tissue from DIO, DIO+BDCM (1w), Lep KO (1w), Lepr KO (1w), MCS (4w), MCD (4w), miR21 KO+BDCM (1w) and miR21 KO+MCD (4w) groups was cut into 5 μm thick sections. Each section was deparaffinized using standard protocol. Briefly, sections were incubated with xylene twice for 3 min, washed with xylene:ethanol (1:1) for 3 min and rehydrated through a series of ethanol (twice with 100%, 95%, 70%, 50%), twice with distilled water and finally rinsed twice with phosphate buffered saline (PBS). Epitope retrieval of deparaffinized sections was carried out using epitope retrieval solution and steamer (IHC-world, Woodstock, MD) following manufacturer’s protocol. The anti-mouse primary antibodies (i) anti-VEGFR-2 was purchased from AbCam Inc. (Cambridge, MA), (ii) anti-ICAM-1, and (iii) anti-E-selectin were purchased from Santa Cruz biotechnology, Inc. (Santa Cruz, CA), and used in 1:150 dilutions. Species-specific anti-IgG secondary antibody conjugated with Alexa Fluor 633 (Invitrogen, California, USA) was used to localize the sinusoidal endothelial dysfunction biomarker proteins. Sections were mounted in ProLong gold antifade reagent with DAPI. Images were taken under 20× objectives using Olympus BX51 microscope.

The distal part of a mouse small intestine and frontal cortex tissues was paraffin embedded and prepared according to the standard protocols and 5 μm thick sections were made. Deparaffinization of the sections were done using the standard protocol. Epitope retrieval of the tissue sections was performed using an epitope retrieval solution and steamer (IHC World, Woodstock, MD, USA). Peroxidase blocking was done using 3% hydrogen peroxide (H₂O₂). Blocking was done with 10% serum followed by overnight incubation at 4 °C with primary antibodies against HMGB1, IL-6, IL-1β, and BDNF at 1:200 dilutions. Species specific biotin conjugated secondary antibodies and streptavidin conjugated horseradish peroxidase (HRP) at 1:500 dilutions were used for immunohistochemistry. 3,3’-diaminobenzidine (DAB) (Sigma-Aldrich, St Louis, USA) was used as a chromogenic substrate. Counterstaining was done using Mayer’s hematoxylin solution (Sigma-Aldrich). The stained sections were mounted with Aqua Mount (Lerner Laboratories, Kalamazoo, MI, USA). Sections were observed using the Olympus BX43 and BX63 microscope (Olympus, USA). Morphometric analysis of the images was done using the Cellsens software from Olympus (Center Valley, PA, USA).