Goat anti mouse igg

The following reagent kits were used: triglyceride test kit (GPO-PAP method) (A035 & 2019007, Changchun Huili Biotechnology Co., Ltd.), high-density lipoprotein cholesterol (direct method-selective inhibition method) (A029 & 2019011, Changchun Huili Biotechnology Co., Ltd.), low-density lipoprotein cholesterol (direct method) (A028 & 2019011 Changchun Huili Biotechnology Co., Ltd.), total cholesterol (A111-1 & 20190515, Nanjing Jiancheng Bioengineering Research Institute), Novostart SYBR qPCR Supermix plus (E096-01B & 0516511, Novoprotein), Primescript™ RT reagent kit with gDNA eraser (RR047A & AJ51485A, TaKaRa), rat high-sensitivity C-reactive protein ELISA Kit (JYM0253Ra & GR2020-06, Wuhan ColorfulGene Technology Co., Ltd.), rat prostaglandin E2 (PGE2) ELISA Kit (JYM0253Ra & GR2020-06, Wuhan ColorfulGene Technology Co., Ltd.), rat leptin ELISA kit (JYM0253Ra & GR2020-06, Wuhan ColorfulGene Technology Co., Ltd.), ECL hypersensitive luminescence kit (34094 & UE283595A, Thermo), goat anti-mouse IgG (ZB-2305 & 140193, Zsbio), and goat anti-rabbit IgG (ZB-2301 & 206820103, Zsbio). The primers were synthesized by SANGON Biotechnology.

IHC was used to verify NLRP3 and IBA1 (a microglial marker in the brain) expression in the ipsilateral cortex. The deparaffinized and rehydrated coronal brain slices (4 μm thick) were prepared as described above. The slices were incubated with 3% H₂O₂ for 10 min at room temperature to quench any endogenous peroxidase activity, and then blocked with 5% goat serum for 20 min at room temperature. The slices were then incubated overnight at 4°C with the following primary antibodies: rabbit anti-NLRP3 (diluted 1:50; NBP1-77080SS, Novus) and mouse anti-IBA1 (diluted 1:100; GB12105, Servicebio). The next day, the brain slices were washed with PBS and incubated for 20 min at room temperature with biotinylated goat anti-rabbit IgG (diluted 1:100; ZSGB-Bio, Beijing, China) or goat anti-mouse IgG (diluted 1:100; ZSGB-Bio). The brain slices were then incubated with horseradish peroxidase (HRP)–streptavidin reagent for 10 min and stained with 3,3′-diaminobenzidine peroxidase substrate. Images were obtained with a light microscope (Leica-DM2500, Wetzlar).

In order to detect proteins related to ischemic brain injury, western blot was used. Briefly, 400 μg of total protein was extracted and dissolved by 15% SDS-polyacrylamide gel. Afterward, the protein was separated to protein bands at 80 voltage (V) sustaining 20 min and then 120 V sustaining 30 min by using the electrophoresis buffer (24.8 mM Tris, 192 mM glycine, and 0.1% SDS). Then, the transfer buffer (24.8 mM Tris, 192 mM glycine, and 10% methanol) was added, the protein on the SDS-PAGE gel was transferred to PVDF membranes for 4 h at a rate of 350 mA. The PVDF membranes were blocked by 5% skim milk at room temperature for 1–2 h, and were then incubated with primary antibody (Beclin-1, 1:2000, ABclonal, USA; LC3I, 1:2000, ABclonal, USA; LC3II, 1:2000, ABclonal, USA, P62, 1: 2000, ABclonal, USA; ATG12, 1:2000, ABclonal, USA) in 3% BSA overnight at 4 °C. After washing in TBST 3 times for 8 min each time, the PVDF was incubated in the secondary antibody (goat anti-rabbit IgG and goat anti-mouse IgG; ZSGB-BIO, Beijing, China, 1:5000) for 1~2 h at room temperature. Finally, the PVDF membranes were rinsed in TBST three times to get ECL luminescence, scanning, and image acquisition using Alpha Ease FC Gel Imaging System (BIO-RAD, USA) with ECL.

The western blotting analysis was performed as previously described [30 (link)]. In brief, cells were lysed by RIPA buffer containing protease inhibitors and phosphatase inhibitors (Pierce, USA). The protein concentration was measured by a BCA regent kit (Pierce, USA). The lysates were loaded into 10% of sodium dodecyl sulfate-polyacrylamide electrophoresis gels and then transferred to nitrocellulose (NC) membranes (PALL, USA). The membrane was blocked with 5% of bovine serum albumin (BSA) in Tris-buffered saline-Tween 20 and then incubated with a primary antibody at 4°C for overnight. The anti-Flag (F3165) was purchased from Sigma. The β-catenin (sc-7199), TGIF (sc-9084), p21 (sc-397), STIM1 (sc-68897), p53 (sc-6243), Akt (sc-8312), p-Akt (sc-33437), cyclin A (sc-751), cyclin B1 (sc-752), cyclin D1 (sc-718), CDK4 (sc-260), and β-actin (sc-8432) were purchased from Santa Cruz Biotechnology. The Rb (#9313S), phospho-Rb (#8516S), ERK1/2 (#4695S), p-ERK1/2 (#4370S), and PTEN (#9188S) were purchased from Cell Signaling Technology. The secondary antibodies (Peroxidase-Conjugated AffiniPure Goat Anti-Rabbit-IgG and Goat Anti-Mouse-IgG) were purchased from ZSGB-BIO (Beijing, China). The signals were detected in the ChemiDoc™ XRS+ Imaging System (Bio-Rad, USA) by a Bio-Rad Clarity™ western ECL substrate (Bio-Rad, USA).

Total proteins were extracted from backfat using radio immunoprecipitation assay lysis buffer (Beyotime, Shanghai, China) and protein content was measured using the bicinchoninic acid protein assay kit (Beyotime, China). Total protein (5 to 20 μL) was loaded onto a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel, separated by electrophoresis, and transferred onto a polyvinylidene difluoride membrane. Blots were blocked with 5% skim milk overnight at 4°C and blotted with specific primary antibodies for LEPR (1:300, bs-20498R, Bioss, Beijing, China) and β-actin (1:2,000, TA-09, ZSGB-BIO, Beijing, China). The bound primary antibodies were determined with a horseradish peroxidase conjugated secondary antibody goat anti-rabbit immunoglobulin G (IgG) (1:2,000, ZB2301, Zsbio, Beijing, China) and a horseradish peroxidase conjugated secondary antibody goat anti-mouse IgG (1:2,000, ZB2305, Zsbio, China), respectively. The data were analyzed using Image J software (GEL system, Beijing kcrx bio-company, Beijing, China). The relative expression of LEPR was calculated as the ration of optical density of LEPR to that of β-actin.

The Western blot experiment was carried out as previously described (Liu et al., 2014 (link)). In brief, tissues from the brain cortex were dissociated and homogenized by radioimmunoprecipitation assay (RIPA) lysis buffer containing protease inhibitor cocktail (Beyotime, Jiangsu, China). A volume of about 80 μg proteins was resolved in 15% sodium dodecyl sulfate (SDS)-polyacrylamide gel (Beijing Junyi Electrophoresis Co., Ltd., Beijing, China) with electrophoresis buffer at 60 V for 30 min, then 100 V for 90 min. The proteins bands were transferred to a polyvinylidenedifluoride (PVDF) membrane with 300 mA for 40 min. For blocking, the membrane was incubated in 5% skim milk at room temperature for 1 h and then was incubated with the primary antibody of PDXK (mouse, Abcam, 1:2,000) and β-actin (mouse, Abcam, 1:5,000) at 4°C overnight. The next day, the membrane was rinsed with 1 × TBS containing 0.2% Tween-20 (TBST; Shanghai Double-Helix Biotech Co., Ltd., Shanghai, China) for four times. Then, the membrane was incubated with 1:5,000 secondary antibody (goat antimouse IgG, ZSGB-BIO, China). Finally, the membrane was rinsed in TBST for four times, and the immune complexes were developed with enhanced chemiluminescence reagent and visualized by ChemiDoc XRS System with Image Lab Software 2.0 (Bio-Rad Laboratories, Inc., Hercules, CA, United States).