Horseradish peroxidase labeled secondary antibody

If appropriate, for brain tissues, four-micron frozen brain sections that were fixed in 4% formaldehyde or acetone were blocked for endogenous peroxidase activity with 0.03% H₂O₂. PHNs were cultured on glass coverslips that were precoated with poly-L-lysine (Sigma-Aldrich) and then were fixed with 4% paraformaldehyde for 30 min and then treated with 0.1% Triton X-100 at room temperature for 10 min. Blocking was achieved with PBS that contained 5% normal goat serum at room temperature for 1 h.
Sections were then incubated at 4°C overnight with the following primary antibodies: IGF-I (monoclonal, 1:200), pERK1/2 (monoclonal, 1:400), pnNOS Ser1417 (monoclonal, 1:200), MAP2 (monoclonal, 1:500; Abcam), pRSK (monoclonal, 1:400; Biorbyt). Binding of primary antibodies was revealed by incubating the sections for 30 min with Alexa Fluor 488 (green)/Alexa Fluor 594 (red)-conjugated secondary antibodies (Abcam) or with horseradish peroxidase-labeled secondary antibodies (Abcam). They were then visualized by diaminobenzidine.

Eicosapentaenoic Acid (purity, ≥97%), 3-Methyladenine (3-MA, a PI3K inhibitor) and Dorsomorphin (Compound C, an AMPK inhibitor) were purchased from MedChemExpress (NJ, United States). Type II collagenase, TBHP, and dimethylsulfoxide (DMSO) were purchased from Sigma-Aldrich (St Louis, MO, United States). Primary antibodies for cleaved-caspase 3 (C-C3), Beclin-1, LC3B, p-mTORC1, mTORC1, BCL-2, AMPK and p-AMPK were procured from Cell Signaling Technologies (Danvers, MA, United States). Antibodies against BAX, BCL-2, Collagen II, MMP13 and P62 were purchased from Abcam (Cambridge, United Kindom). Antibodies against p-PERK, PERK, p-eIF2α, eIF2α, CHOP, ADAMTS5, Aggrecan, and ATG5 were purchased from ABclonal (WH, CHINA). Antibodies against ATF4, GRP78, Collagen II, and β-actin were purchased from Proteintech (NJ, China). Horseradish peroxidase-labeled secondary antibodies, Alexa Fluor^® 488-labeled goat anti-rabbit IgG (H + L) secondary antibody, and Alexa Fluor^® 594-labeled goat anti-mouse IgG (H + L) secondary antibody were purchased from Abcam. Further, 4′,6-diamidino-2-phenylindole (DAPI) was purchased from Beyotime (SH, CHINA). The reagents for cell culture were obtained from Gibco (Grand Island, NY, United States).

The samples were lysed in RIPA buffer (CST, Boston, MA, USA) and the proteins were quantified using the bicinchoninic acid method (Thermofisher, Waltham, MA, USA). The lysates were centrifuged (10,000 rcf, 5min), subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to polyvinylidene fluoride membranes. Subsequently, the membranes were blocked and incubated with the specific primary antibody overnight at 4 °C, followed by horseradish peroxidase-labeled secondary antibodies (dilution 1:5000–10,000; Abcam, Cambridge, UK). The protein expression was visualized using enhanced chemiluminescence reagents (Amersham, Piscataway, NJ, USA) and the ChemiDoc XRS system (Bio-Rad, Hercules, CA, USA). The primary antibodies used are as follows: 3-hydroxy-3-methyl glutaryl coenzyme A reductases (HMGCR; # sc-271595; Santa Cruz Biotechnology, Santa Cruz, CA, USA), fatty acid synthetase (FAS; #ab133619; Abcam, Cambridge, UK), sterol regulatory element binding protein 2 (SREBP2; #ab30682 Abcam, Cambridge, UK), 1-acyl-sn-glycerol-3-phosphate acyltransferase-beta-1 (Agpat1; #bs-5023R; Bioss, Beijing, China), and diacylglycerol-acyltransferase-1 (Dgat1; #sc-271934; Santa Cruz Biotechnology, Santa Cruz, CA, USA). The antibody for GAPDH (#9485, 1:2500; Abcam, Cambridge, UK) was used as a control.

Ficoll-Paque PREMIUM was obtained from GE Healthcare (Buckinghamshire, UK). Melatonin, chloroquine (CQ), and dimethylsulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Melatonin was dissolved in DMSO as a 200 mM stock solution and stored at −20 °C. Further dilution was performed in cell culture medium. Bovine serum albumin (BSA) and AGE-BSA (AGEs) were obtained from Merck-Millipore (Darmstadt, Germany). Compound C was purchased from Selleckchem (Houston, TX, USA). Primary antibodies against LC3, beclin-1, cleaved caspase-3, Bax, Bcl-2, caspase-9, and cytochrome c were obtained from Cell Signaling Technologies (Beverly, MA, USA). Primary antibodies against p-mTOR, mTOR, p-4EBP1, 4EBP1, p-AMPKα, AMPKα, p-p70S6K, p70S6K, and P62 and fluorescein isothiocyanate (FITC)-labeled and horseradish peroxidase-labeled secondary antibodies were purchased from Abcam (Cambridge, UK). Crystal violet and 4′,6-diamidino-2-phenylindole (DAPI) were obtained from Beyotime (Shanghai, China).

Metformin, 3-methyladenine (3-MA), TBHP and the type II collagenases were from Sigma-Aldrich (St Louis, MO, USA). The primary antibody of p-AMPKα, AMPKα, p16INK4α, P62 and β-actin were acquired from Abcam (Cambridge, UK). The LC-3, Beclin-1, atg3, atg7, atg12, p-p53, p53, p21, cleaved caspase3, Bax and Bcl-2 antibodies were obtained from CST (MA, USA). The fluorescein isothiocyanate-labeled and horseradish peroxidase-labeled secondary antibodies were purchased from Abcam. The 4', 6-diamidino-2-phenylindole (DAPI) was obtained from Beyotime (Shanghai, China). The cell culture reagents were purchased from Gibco (Grand Island, NY, USA).

The proteins from cells were extracted and the protein concentrations were determined referring to the instructions of the bicinchoninic acid assay kits (Beyotime Institute of Biotechnology, Shanghai, China). The proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. Subsequently, the protein samples were transferred to polyvinylidene fluoride membranes and blocked with 5% bovine serum albumin at 4°C. Afterwards, the membranes were added with the primary antibodies to SP1 (1 : 500), Caspase-3 (1 : 500), Bax (1 : 1000), Bcl-2 (1 : 500) (Abcam, Cambridge, MA, USA) and GAPDH (1: 1000) (Millipore Corporation, Bedford, CA, USA) and incubated at 4°C overnight. The membranes were then rinsed with Tris-buffered saline and Tween 3 times. The membranes were incubated with corresponding horseradish peroxidase-labeled secondary antibodies (Abcam, Cambridge, MA, USA) for 1 h at 37°C. An enhanced chemiluminescent solution was used for developing. The gray value of the target band was analyzed by Image J software.