Ab65390

Blood samples were collected by puncturing of the V. maxillaris of anesthetized mice. After clotting, blood samples were centrifuged at 2000× g for 10 min. Resulting supernatant (serum) was transferred into clean tubes and stored at −80 °C until use. Measurement of serum albumin was performed using a mouse-specific albumin ELISA kit (ab108792, Abcam). Creatinine and urea measurements were performed using enzymatic kits (Creatinine PAP LT-SYS LT-CR 0106, Urea LT-UR 0010, Labor & Technik, Eberhard Lehmann GmbH). Serum triglyceride (TG) levels were assayed using a fluorometric kit (ab178780, Abcam). Total cholesterol (TC), high-density lipoprotein (HDL), very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) fractions were measured using a calorimetric kit (ab65390, Abcam). Endotoxin (LPS) levels were determined using a chromogenic endotoxin quantification kit (A39552S, Thermo Fisher Scientific, Inc.). All assays were used according to the manufacturer´s instructions.

At baseline and the end of weeks 4 and 8, blood samples were collected from the facial vein of conscious mice [23 (link)]. Blood serum was obtained after centrifugation (500 g for 10 min at 4°C) and stored at −20°C until being used to assess blood lipid profiles (total cholesterol (TC), high density lipoprotein (HDL), low/very low density lipoprotein (LDL/VLDL), and cholesterol) using a commercially available ELISA assay kit (ab65390, ABCAM, UK) according to the manufacturer's directions.

Blood serum was obtained by centrifugation (500 g for 10 min at 4°C) and stored at -20°C until being used to assess blood lipid profiles (total cholesterol (TC), high density lipoprotein (HDL), low/very low density lipoprotein (LDL/VLDL) and cholesterol) using a commercially available ELISA assay kit (ab65390, ABCAM, U.K.) according to the manufacturer’s directions.
The serum testosterone, FSH and LH levels were assessed using the total antioxidant capacity kit (ab65329, Abcam, United Kingdom), testosterone ELISA kit (ab108666, Abcam, United Kingdom), FSH ELISA kit (MBS703380, MyBioSource, U.S.A) and LH ELISA kit (MBS041300, MyBioSource, U.S.A), respectively (according to the manufacturers protocols).

Tissue glycogen (10 mg tissue, Abcam #ab65620) and cholesterol (20 mg tissue, Abcam #ab65390) were measured according to the manufacturer's instructions. For protein extraction and quantification, 40 mg tissue samples were homogenized in 1 ml of prepared RIPA buffer with protease inhibitor (Roche #COUEDTAF-RO) and phosphatase inhibitor (Roche #PHOSS-RO) on ice. After centrifugation at 4°C, 13000× g for 20 min, the supernatant was aliquoted and quantified by the BCA method. For RNA/DNA extraction and quantification, 60–80 mg tissue samples were homogenized in 1 ml TRIzol reagent (Invitrogen #15596026) and extracted for RNA/DNA according to the manufacturer's instructions (Xin et al., 2021a (link)). RNA/DNA concentration was measured in a spectrometer (Thermo, NanoDrop 2000C). Concentrations of these tissue biochemicals were normalized to wet weight of liver tissues.

The levels of circulating free fatty acids (ab65341), triglyceride (ab65336), total cholesterol, low-density lipoprotein (LDL), and high-density lipoprotein (HDL) (ab65390), all from Abcam, Cambridge, MA, USA, and oxidized low-density lipoprotein (oxLDL) (MBS2512757, MyBioSource, San Diego, CA, USA) were measured according to the manufacturer's instructions.

According to the manufacturer’s instructions, plasma immunoglobulin (Ig) A, G, and M levels were measured using commercial ELISA kits (Shanghai Jianglai Biotechnology Co., Ltd., Shanghai, China). The assays limits of detection, limits of quantification, intra-assay CV%, and interassay CV% are presented in Table 3 according to the manufacturer-provided data.
Plasma corticosterone level was quantified using chicken-specific quantitative competitive ELISA kit (MBS701668; MyBioSource, San Diego, CA, USA). According to the manufacturer, the CV% for the intra- and interassay were <8% and <10%, respectively, with a detection range of 0.5–20 ng/mL. Meanwhile, plasma total protein (TP), triglyceride (TG), and total cholesterol levels were quantified according to the kits’ protocols using quantitative colorimetric assays (ab102535, ab65336, and ab65390, respectively; Abcam, Waltham, MA, USA). As for liver enzyme activity, alanine amino transferase (ALT) and aspartate amino transferase (AST) activities were determined using commercial kits applying enzyme activity colorimetric assays (ab241035 and ab105135, respectively; Abcam, Waltham, MA, USA).