Ab26278

Formalin-fixed and paraffin-embedded rats’ pancreatic tissue sections were deparaffinized in xylene and rehydrated using graded ethanol into PBS. Endogenous peroxidase was blocked with 0.3% hydrogen peroxide in methanol. The sections were stained with H&E pending histopathological analysis. For the protein content and distribution, the sections were applied for IHC staining. Sections were incubated with 5% normal rat serum followed by further incubation; firstly, with primary antibodies against ABCA1 (ab18180, Abcam) and GLP-1 (ab26278, Abcam) at 4°C overnight, and secondly, with a biotinylated secondary antibody, and thirdly, with an avidin-biotinylated peroxidase complex (Santa Cruz Biotechnology). The HE and IHC staining sections were observed and determined through a double-blind study by two authors. Cytoplasmic staining for ABCA1 or GLP-1 was considered positive. Five random visual fields from each section were selected and analyzed under 400× magnification. The positive area were calculated, which is showed in percentage (ratio of positive area to the whole visual field).

Livers were harvested from sacrificed rats and fixed in 4% paraformaldehyde, dehydrated, followed by paraffin embedding and sectioning at 5 mm thickness. The sections were deparaffinized in xylene and rehydrated in a graded series of ethanol and then subjected to H&E or immunohistochemistry (IHC) staining. For H&E staining, the slides were rehydrated with descending gradient of ethanol, stained with hematoxylin and eosin, respectively, dehydrated with ascending gradient of ethanol, and then cleared with xylene. IHC staining was performed with Vectastain Elite ABC Kit (Vector Laboratories) following the manufacturer's instructions. Citrate buffer (pH 6.0) was used for antigen retrieval, and 0.3% NaHB₄ was used for immunoperoxidase labeling. Sections were incubated with primary antibody (anti‐GLP1 (Affinity AF0166; Abcam, ab26278), anti‐Ghrelin (Abcam, ab209790)) at 4 °C overnight. Immunoreactive sites were visualized with 3, 30‐DAB and the resulting signals were visualized using a confocal laser scanning microscope (Olympus BX61, Tokyo, Japan).
Evaluation of steatosis in the sections was performed by two experienced pathologists using the NAFLD histological scoring system where the degree of steatosis was graded into four levels. A score of 0 was assigned for <5% steatosis, 1 for 5–33% steatosis, 2 for 34–66% steatosis, and 3 for >66% steatosis.^[55]

Segments of distal ileum (1 cm of intestine withdrawn above the junction with the caecum) were harvested from all rats and fixed in 10% neutral-buffered formalin for 24 h, dehydrated and embedded in paraffin. The paraffin-embedded tissue sections were cut (3 μm thickness) and mounted on adhesive microscope slides. The sections were deparaffinized in xylene and rehydrated with decreasing concentrations of ethanol (100%, 95% and 70%) and distilled water. Antigen retrieval was performed in 0.01 M sodium citrate buffer with 0.05% Tween 20 (pH 6.0), using a water bath at 98 °C for 10 min and blocked in 3% H₂O₂ in PBS. After that, sections were incubated with a blocking solution (Rodent Block M, RBM961G, Biocare Medical, UK) for 30 minutes and then incubated overnight at 4 °C with anti-GLP-1 primary antibody: mouse monoclonal, ab26278; Abcam, Amsterdam, The Netherlands; 1:1500. Subsequently, sections were incubated with secondary antibody at RT for 30 min: goat anti-mouse, ab97021; Abcam, Amsterdam, The Netherlands; 1:200. Then, 30-minute ABC complex incubation was carried out (Vector A and B, Vectastain, Vector Labs, Peterborough, UK). Finally, the sections were treated with diaminobenzidine tetrahydrochloride (DAB) substrate kit (ab64238, Abcam, Amsterdam, The Netherlands). All sections were stained with H&E.