Goat anti mouse igg h l alexa fluor 647 secondary antibody

HEK293T/17 cells were transfected with pEVAC encoding representative NA sequences of N1, N2, N3, N4, N8 and N9, as per PV production. Forty-eight hours post-transfection, cells (50,000 cells/well) were transferred into V-bottom 96-well plates. Cells were then incubated with mouse sera (diluted 1:50 in PBS) for 30 min, washed with fluorescence-activated cell sorting (FACS) buffer (PBS, 1% (v/v) FBS, 0.02% (v/v) Tween 20) and stained with goat anti-mouse IgG (H+L) Alexa Fluor 647 Secondary Antibody (Thermo Fisher A-21235, Paisley, UK) diluted at 20 µg/mL in FACS buffer for 30 min in the dark. Cells were washed with FACS buffer, and samples were run on the Attune NxT Flow Cytometer (Invitrogen) with a high throughput autosampler. Dead cells were excluded from the analysis by staining cells with 7-aminoactinomycin D (7-AAD) and gating 7-AAD negative cells.

PCLS from Vangl2^flox/flox, Vangl2^Lp/+ and wild-type mice were fixed with 4% (v/v) paraformaldehyde (PFA) for 15 min at room temperature (RT), washed three times in PBS, permeabilised with 0.5% Triton X-100 in PBS at RT for 30 min, followed by 1 h blocking with PBSBT (1% bovine serum albumin, 0.2% Triton X-100 in PBS) at RT. After blocking, PCLS were incubated with mouse anti-pan-cytokeratin (Sigma-Aldrich, C2931; 1:200) diluted in PBSBT blocking buffer at 4°C overnight. After three washes in PBSBT, PCLS were then incubated with Rhodamine Phalloidin (Biotium, 00027; 1:200) and goat anti-mouse IgG (H+L) Alexa Fluor 647 secondary antibody (Thermo Fisher Scientific, 21235; 1:500) at RT for 2 h (antibody details are provided in Table S1). After washing in PBS, cell nuclei were labelled with DAPI (Sigma Aldrich, D9542; 1:500). Coverslips were then mounted with ProLong™ Gold Antifade Mountant (Invitrogen, P36930). PCLS were imaged on a Leica SP8 inverted confocal microscope using an HC PL APO 40×/1.30 oil objective lens. For some images, channel colours were changed during image post-processing for optimal data visualisation.

hMSC characterization by flow cytometry was performed on cells before seeding on biomaterials to demonstrate their characteristics as defined by the ISCT criteria (Dominici et al., 2006 (link)). hMSC were stained with CD90-FITC, CD73-PE, CD105-PC7, CD45-APC-A750 antibodies (Beckman Coulter, Brea, California, United States), or corresponding isotype controls in matched concentration, 20 min at room temperature (RT) and in the dark. Cells were washed in PBS without Ca²⁺/Mg²⁺ (Thermo Fisher), fixed, and permeabilized (IntraPrep Permeabilization Kit, Beckman Coulter). Intracellular staining was performed by an indirect immunofluorescence method with an adapted dilution of Nestin antibody (Merck Millipore, Burlington, Massachusetts, United States), or its corresponding isotype control in matched concentration, and with an Alexa Fluor 647 goat anti-mouse IgG (H + L) secondary antibody (Thermo Fisher). After washing, cells were analyzed with a NAVIOS flow cytometer (Beckman Coulter) and data files were interpretated using Kaluza software (Beckman Coulter).