Goat anti mouse igg h l antibody conjugated with alexa fluor 488

The monoclonal antibody against the PRRSV N protein was a kind present from Dr. Ying Fang (Department of Animal Sciences and Industry, Kansas State University, Manhattan). Monoclonal antibodies against PRRSV nsp2, PCSK9, and CD163 were produced and stored by our lab. Goat anti-mouse IgG (H + L) antibody conjugated with Alexa Fluor 488 (1:800) was purchased from Abcam (Abcam, Shanghai, China). MG132 and CQ were purchased from Sigma (Sigma, Shanghai, China). DMSO was purchased from MP Biomedicals (MP Biomedicals, Shanghai, China). Lipofectamine^®3000 Transfection Kits were purchased from Invitrogen (Invitrogen, Shanghai, China). Dual Luciferase Reporter Assay Kits were purchased from Vazyme Biotechnology (Vazyme Biotechnology, Nanjing, China). Monoclonal Anti-HA-Agarose antibody produced in mice was purchased from Sigma (Sigma, Shanghai, China). PrimeSTAR^® HS DNA Polymerase with GC Buffer was purchased from Takara (Takara, Dalian, China). DAPI was purchased from Beyotime (Beyotime, Shanghai, China).

The cells were cultured on Cur_WPI and PS as described above (Section 2.5.4). After 12 days of culture, collagen type I, collagen type II, aggrecan, and SOX-9 were visualized via immunofluorescence staining. Thus, the cells were rinsed with PBS, fixed with 3.7% paraformaldehyde, permeabilized with 0.2% Triton^TMX-100, and blocked with 1% bovine serum albumin solution (all reagents from Sigma-Aldrich, Warsaw, Poland). Then, the cells were stained overnight with 10 μg/mL of the following primary antibodies: rabbit polyclonal anti-collagen I antibody (Invitrogen, ThermoFisher Scientific, Waltham, MA, USA), rabbit polyclonal anti-collagen II antibody (Abcam, Cambridge, UK), mouse monoclonal anti-aggrecan antibody (Invitrogen, ThermoFisher Scientific, Waltham, MA, USA), and rabbit monoclonal anti-SOX-9 antibody (Abcam, Cambridge, UK). Subsequently, the cells were stained with 2 μg/mL of secondary antibodies, namely goat anti-rabbit IgG (H + L) antibody-conjugated with AlexaFluor^® 488 or goat anti-mouse IgG (H + L) antibody-conjugated with AlexaFluor^® 488 (both reagents from Abcam, Cambridge, UK). Additionally, cell nuclei were stained with Hoechst 33342 (Sigma-Aldrich, Warsaw, Poland). The cells were observed using a CLSM (Olympus Fluoview equipped with FV1000, Shinjuku, Japan). Nuclei emitted a blue fluorescence, while evaluated markers emitted a green one.