Mouse ins insulin elisa kit

After being gradient dehydrated by 20% and 30% glucose, brains were embedded with OCT glue and frozen in a cryostat (Cryostar NX50™, Thermo Scientific) for over 1 h. The frozen brain was prepared into 16 μm thick sections, then incubated with goat serum containing 0.2% Trion-100 at 37 °C for 30 min. NDRG2 labeling was performed by incubating with primary anti-NDRG2 rabbit monoclonal antibody (ab174850-IF, Abcam, UK), anti-GFAP chicken polyclonal antibody (GTX85454, Gene Tex, USA), and anti-NeuN mouse monoclonal antibody (MAB377, Merck Millipore, USA) overnight at 4 °C. The sections were then washed with PBS (phosphate-buffered saline) for 7 min × 3 times. The next day, sections were incubated for 2 h with fluorescent secondary antibodies Alexa Fluor®488 (139424, Jackson Immuno, USA), Alexa Fluor® 594 (ab150064, Abcam, UK), and Alexa Fluor® 647 (ab150115, Abcam, UK). DAPI was added during the final 5 min of incubation. The sections were stored at 4 °C until observed, and images were captured as soon as possible using a confocal fluorescence microscope (BX51, Olympus, Tokyo, Japan) or super-resolution confocal microscope (Leica TCS SP8 STED 3X). Insulin concentration was detected according to the instructions of a Mouse INS (Insulin) ELISA Kit (E-EL-M1382c) purchased from Elabscience, Wuhan, China.

After 12 weeks of intervention by Semaglutide, mice were fasted for 12 h, and tail blood was taken to measure fasting blood glucose with a glucose meter (Accu-Chek; Roche Diagnostics GmbH, Germany). Mouse INS (Insulin) ELISA Kit (E-EL-M1382c, Elabscience Biotechnology Co., Ltd, China) was used to determine the level of Insulin. Fasting Insulin Resistance index (HOMA-IR) was also assessed: HOMA-IR=fasting glucose×fasting insulin/22.5. Then, the mice were anesthetized with 40 mg/kg pentobarbital sodium, and the blood from inner canthus was collected and centrifuged at 2000 r/min for 20 min. Finally, the plasma was separated and tested for lipid and inflammatory indexes, including: triglyceride (TG), serum cholesterol (CHO), low-density lipoprotein (LDL), high-density lipoprotein (HDL), tumour necrosis factor-ɑ (TNF-α), interleukin-6 (IL-6), and IL-1β. TG and CHO were detected by the GPO-PAP method (NanJing JianCheng Bioengineering Institute, China). LDL and HDL were measured by the terminal method. Mouse IL-1β ELISA Kit (70-EK201B/3-96, MultiSciences Biotech Co., Ltd., China), Mouse IL-6 ELISA Kit (70-EK206/3-96, MultiSciences Biotech Co., Ltd., China) and Mouse TNF-α ELISA Kit (70-EK282/4-96, MultiSciences Biotech Co., Ltd., China) was used to determine the level of IL-1β, IL-6 and TNF-α, respectively.