Alexa fluor 488 conjugated goat anti mouse igg h l antibody

Minimal essential medium (MEM), F12 medium, fetal bovine serum (FBS), non-essential amino acids and antibiotic-antimycotic (100X), Trizol reagent, B27 supplement, Neurobasal medium, Lipofectamine 2000 Transfection Reagent and Alexa Fluor 488-conjugated goat anti-mouse IgG (H+L) antibody were purchased from Invitrogen (Carlsbad, CA, USA). Fluo-4 AM, MitoSOX, Mitotracker green, Hoechst 33,342 stain and 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) were purchased from Molecular Probes (Chicago, IL, USA). NMDA and D(−)-2-Amino-5-phosphonopentanoic acid (APV) were obtained from Tocris (Bristol, UK). Monoclonal Glur1/Nr1 antibody was obtained from Neuromab Antibodies Inc., (Davis, CA, USA). ASX- rich pigment (Supplementary Figure S1) was extracted from Lithodes antarcticus (supplementary methods; BIOTEX S.A., Santiago, Chile). pcDNA3-Cyto-GCaMP3 (Plasmid #64853: named in the text also as Gcamp-actin), was acquired from Addgene (Teddington, UK).

MARC-145 and pCD163-MARC cells were seeded directly onto coverslips. 48 h later, the expression of pCD163 was identified by IFA. IFA was also conducted to confirm the N protein expression of the 5th-passage PRRSV isolated from clinical specimen. Briefly, cells were washed twice in ice-cold phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde in PBS at 4°C for 1 h. Cells were then washed three times with ice-cold PBS and permeabilized with 0.5% Triton X-100 for 15 min. The coverslips were then incubated with mouse anti-pCD163 MAb (1 : 500) or anti-N MAb SDOW17 (Rural Technologies, Brookings, SD, USA) for 1 h. After three washes with PBS, coverslips were incubated with Alexa Fluor 488-conjugated goat anti-mouse IgG(H+L) antibody (Invitrogen) at room temperature for 1 h. Then the coverslips were washed three times and mounted with mounting buffer (60% glycerol and 0.1% sodium azide in PBS) and observed under an Olympus BX51 inverted fluorescence microscope.

Reagents were from the following suppliers: LPA (L7260), DMSO (D2650), BSA (A6003), propidium iodide (PI, P4170) (Sigma, UK); human-EGF (PHG0313; Gibco); Ki16425 (S1315), batimastat (BB94, S1715), AG1478 (S2728), LY294002 (S1105), rapamycin (S1039), CCT137690 (S2744) (Selleck); 4’,6-diamidino-2-phenylindole (DAPI, D3571) (Invitrogen, Carlsbad, CA, United States). Antibodies were from the following suppliers: anti-phosphotyrosine (EPR16871; Abcam); anti-GAPDH (HC301), anti-β-actin (HC201), anti-β-tubulin (HC101), anti-rabbit (HS101), and anti-mouse (HS201) antibodies (TransGen Biotech, Beijing, China); anti-Bax (bs0127R), anti-Bcl 2 (bs0032R) antibodies (Bioss, Beijing, China); anti-BrdU (abs128684), anti-AURKA (abs136365), anti-phospho-Aurora kinase (Thr288) (abs130639) antibodies (Absin, Shanghai, China); anti-caspase 3/p17 (19677-1-AP), anti-caspase 6/p18/p11 (10198-1-AP), anti-caspase7 (27155-1-AP), anti-caspase 9 (10380-1-AP), anti-PARP (13371-1-AP), anti-EGFR (18986-1-AP), anti-geminin (10802-1-AP) antibodies (Proteintech Group, Wuhan, Hubei, China); anti-LC3B (AF4650, Affinity Biosciences, United States); Alexa Fluor 488–conjugated goat anti-mouse IgG (H + L) antibody (A11008, Invitrogen).

Mycoplasmas were labeled with carboxyfluorescein diacetate succinimidyl ester (CFDA-SE, Beyotime Biotechnology, Shanghai, China) for 30 min at 37 °C. EBL cell monolayers were then incubated with CFDA-SE labeled mycoplasmas in 1 mL at a multiplicity of infection (MOI) of 10³. For protein binding assays, EBL cell monolayers were incubated with 10 µg of rMbovP0503, either alone or preincubated with 10 µL of anti-rMbovP0503 sera or normal mouse serum as control. After 1 h incubation at 37 °C, cell monolayers were washed with DPBS, fixed with 4% neutralized paraformaldehyde for 30 min, treated with 0.5% Triton X-100 for 5 min, and blocked with 5% BSA for 2 h at 37 °C. Actin filaments and nuclei were labeled at RT with 200 mM Rhodamine phalloidin (Cytoskeleton, Denver, CO, USA) and 100 nM 4, 6-diamidino-2-phenylindole (DAPI), respectively. The binding of rMbovP0503 was visualized using rMbovP0503 mouse antiserum and Alexa Fluor^® 488-conjugated goat antimouse IgG (H+L) antibody (Invitrogen Corporation, Carlsbad, CA, USA). Immunofluorescence was detected with an Olympus FV1000 laser scanning confocal microscope (Olympus FV1000 and IX81, Tokyo, Japan). The experiments were repeated three times independently.

MDCK cells were seeded in a 96-well plate (1 × 10⁴ cells/well). H₂O, EtOAc, Hex, or CHCl₃ extracts from the stems of J. multifida (3.1-25 μg/mL) or (+)-(S)-bakuchiol (3.1-25 μM) were mixed with influenza A virus at a MOI of 0.1 in the infection medium and incubated for 30 min at 37 °C in the presence of 5% CO₂. DMSO (0.031-0.25%) was used as the negative control. Each mixture was added to the cells and incubated for 24 h at 37 °C in the presence of 5% CO₂. The cells were fixed with 4% paraformaldehyde in PBS for 30 min at 4 °C and then permeabilized by the addition of 0.3% Triton X-100 for 20 min at 25 °C. A mouse antibody for the detection of the NP of A/PR/8/34 (FluA-NP 4 F1; SouthernBiotech, AL, USA) was used as the primary antibody. Alexa Fluor488-conjugated goat anti-mouse IgG (H + L) antibody (Life Technologies, CA, USA) was used as the secondary antibody. Cell nuclei were then stained using diamidino-2-phenylindole (DAPI; Life Technologies). The wells were photographed using a fluorescence microscope (BIOREVO BZ-9000, Keyence, Osaka, Japan), and the percentage of influenza A NP-positive cells per DAPI-positive cells were calculated based on measurements recorded with BZ-H1C software (Keyence).