Alexa fluor 488 labeled goat anti rabbit igg h l secondary antibody

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).

HEK-293A cells were seeded on cell culture slide and incubated at 37 °C in a 7% CO₂ incubator to be 70% confluent by the next day. Cells were then transfected with 1 μg of either pVAX-S1 or pVAX control plasmid using Lipofectamine 2000 Transfection Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions and incubated at 37 °C in a 7% CO₂ incubator for 36 h. The media was removed, cells were washed with PBS, fixed with 4% formaldehyde at 4 °C for 10 min and permeabilized with 0.2% PBS-Triton X-100 (PBS-Triton) at 4 °C for 20 min. Cells were washed twice with PBS-Triton and blocked with 2% goat serum/PBS-Triton at room temperature for 30 min. Cells were then stained with in-house rabbit anti-S primary polyclonal antibodies at a 1:1000 dilution in 2% goat serum/PBS-Triton at 4 °C for 1 h. This was followed by three washes and staining with Alexa Fluor-488 labeled goat anti-rabbit IgG H&L secondary antibody (Abcam, UK) at 1:500 dilution in blocking buffer in the dark at room temperature for 1 h. Cells were finally washed three times with PBS-Triton and mounted with VECTASHIELD with DAPI counterstain antifade mounting medium (Vector Laboratories, Inc., Burlingame, CA, USA). Images were captured using Olympus BX51 Fluorescence Microscope and analyzed using ImageJ 1.53e Software.