Au400 automatic biochemical analyzer

First, the calibration and the determination of quality control products for all biochemical markers were performed. After all results of quality control products were within the permitted ranges, the concentrations of K⁺, Na⁺, and Cl⁻ in seminal plasma and serum specimens were detected with a PSD-16a Electrolyte Analyzer and all other biochemical markers determined with Olympus AU400 Automatic Biochemical Analyzer. The samples outside of the linearity of the method and instrumentation were diluted to obtain effective results. All serum samples were directly determined without any dilution, while the measurements of LDH, CK, αHBDH, K⁺, Ca, and Mg in seminal plasma were conducted after 1 : 5 dilution, and 1 : 25 dilution for GGT and P. The seminal plasma samples for the measurements of LDH, CK, αHBDH, Ca, Mg, GGT, and P were diluted with normal saline, and that for K⁺ was diluted with deionized water.

Antibodies used in our experiments included anti-SHH, anti-Gli1, anti-snail1, and anti-GAPDH antibodies (Affinity Biosciences, China). PVDF membrane, BCA Protein Assay Kit, and BeyoECL Moon kit were acquired from Beyotime Biotechnology, China. Cyclopamine was purchased from Selleck Chemicals, USA. Experimental instruments included AU400 automatic biochemical analyzer (OLYMPUS, Japan), BX51T-PHD-J11 microscope (OLYMPUS Company, Japan), image acquisition system CMOS (OLYMPUS Company, Japan), and Image-Pro Plus (Media Cybernetics Company, USA), etc.

The venous blood was collected using a heparin sodium anticoagulation tube 12 hour after fasting in the morning of the next day after admission. Following centrifugation, the levels of fasting plasma glucose (FPG), total glyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), hemoglobin A1c (HbA1c), alanine aminotransferase (ALT), aspartate aminotransferase (AST), serum creatinine (Scr), creatine kinase (CK), creatine kinase-muscle/brain (CK-MB), lactate dehydrogenase (LDH), high-sensitivity C-reactive protein (hs-CRP), blood urea nitrogen (BUN) and uric acid (UA) were measured with Olympus AU400 automatic biochemical analyzer (Japan).

At the end of the experiment, glycated hemoglobin (GHb) was measured using the Glycosylated Hemoglobin (GHb) enzyme linked immunosorbent assay (ELISA) Kit (Wuhan ColorfulGene Biological Technology Co., Ltd., Wuhan, China). Plasma insulin concentration (INS) was analyzed using commercial enzyme-linked immunoassay kit (Qiaoyi Biology, Anhui, China). Homeostasis model assessment of insulin resistance (HOMA-IR) was calculated as [glucose (mmol/L) × insulin (mU/L)]/22.5. Total cholesterol (TC), triglyceride (TG), low density lipoprotein (LDL) and high density lipoprotein (HDL) levels in each group were respectively determined using AU400 automatic biochemical analyzer (Olympus, Japan).

Hippocampal tissue was isolated from 50 mg of brain tissue, and then, RIPA lysis buffer was added (ThermoFisher, Waltham, MA, USA). After full homogenization, the tissue was centrifuged at 12000 rpm for 30 min at 4°C and the supernatant was taken. The levels of malondialdehyde (MDA), myeloperoxidase (MPO), and superoxide dismutase (SOD) activity in the tissue were detected by the AU-400 automatic biochemical analyzer (Olympus, Tokyo, Japan).

To avoid the potential interference of OGTT on hormone levels, the rats were allowed three days for recovery. At day 56, the fasting blood were collected by orbital venous between 9:00 and 11:00. The serum was separated by centrifuged at 4 °C, 4000 rpm for 10 min. The rats were killed by decapitation. The liver tissue was divided into small pieces (100 mg) and immediately frozen in liquid nitrogen and then stored at −80 °C until analysis. A piece of liver tissue was fixed in 10% formalin for preparation of paraffin slice. Another piece of liver tissue was prepared to frozen slice for oil-red O staining. The serum levels of glucose, TG, total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), albumin (ALB), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were determined by AU400 automatic biochemical analyzer (OLYMPUS Co., Ltd, Tokyo, Japan). The serum levels of insulin and glucagon were measured by ELISA kits. The homeostasis model assessment insulin resistance index (HOMA-IR) was concluded as the formula: HOMA-IR = Fasting glucose (mmol/L) × Fasting insulin (mIU/L)/22.5. The hepatic glycogen contents were determined according to the instructions of manufacturer.