Alexa fluor 647 goat anti rabbit igg h l

Naïve HUH7 cells or HUH7 cells expressing ORF1 WT, WT-HA-tag, C483A-HA-tag, C563-HA-tag, D248A-HA-tag, or H249A-HA-tag were seeded onto separate glass coverslips (#1.5; 10 mm; Thomas Scientific, Swedesboro, NJ, USA) in a 24-well plate at 100,000 cells per well. 2 days post seeding, the cells were fixed with 4% PFA for 15 min and subsequently permeabilized in 0.25% Triton x-100 for 15 min. The rabbit anti-HA tag, C29F4 (Cell Signaling Technology, Danvers, MA, USA) primary antibody was used at a ratio of 1:1000 (V/V), and the AlexaFluor647 (goat anti-rabbit IgG [H+L], ThermoFisher Scientific, Waltham, MA, USA) secondary antibody was used at a final concentration of 1 µg/mL. All antibodies were diluted with PBS and incubated for 40 min at room temperature (RT). Hoechst 33342 (ThermoFisher Scientific, Waltham, MA, USA) was incubated at a final concentration of 1 µg/mL for 10 min at RT. The coverslips were then mounted onto glass microscopic slides (VWR International, Radnor, PA, USA) with 5 µL of ProLong gold antifade reagent (ThermoFischer Scientific, Waltham, MA, USA). The stained samples were imaged using the Nikon A1R-Si microscope (Nikon, Melville, NY, USA) in the Princeton University Confocal Microscopy Facility. The images were taken at 40× magnification. Images were then analyzed using Fiji (ImageJ2) image analysis software.

The following primary antibodies were obtained from commercial resources: mouse anti-Flag monoclonal antibody (F1804, Sigma-Aldrich), mouse anti-HA monoclonal antibody (H9658, Sigma-Aldrich), mouse anti-GFP monoclonal antibody (66002-1-lg, Proteintech), mouse anti-β actin monoclonal antibody (A2228, Sigma-Aldrich), rabbit anti-GM130 polyclonal antibody (11308-1-AP, Proteintech), and rabbit anti-BAG3 polyclonal antibody (10599-1-AP, Proteintech). The validations for the Golgi marker can be found in the website (GM130, http://www.ptgcn.com/Products/GOLGA2,GM130-Antibody-11308-1-AP.htm). The mouse anti-PRV gB monoclonal antibody (1E7) was prepared and stored in our laboratory.
The following secondary antibodies were used for Western blot analyses: DyLight 800 labeled goat anti-rabbit IgG (H+L) and DyLight 800 labeled goat anti-mouse IgG (H+L) (KPL, MA, USA) for immunofluorescence analyses: Alexa Fluor 488 goat anti-mouse IgG (H+L), Alexa Fluor 568 goat anti-rabbit or mouse IgG; (H+L), and Alexa Fluor 647 goat anti-rabbit IgG (H+L) (Thermo Fisher Scientific, Beijing, China). The cellular nuclei were stained with 4′,6-Diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific, Beijing, China).

DFBL-18689-V2 was acquired from the Public Repository of Xenografts (PRoXe). PDX cells (0.25–1 × 10⁶) were injected IV into 6–8-week-old NSG mice, and mice were sacrificed on day 26–31 after injection. Single-cell suspensions were obtained from spleens, and erythrocytes were lysed (ACK Lysing Buffer). Cells were stained with combinations of the following antibodies: anti-mouse CD45-APC (Cat#559864, clone 30-F11), anti-mouse CD45.1-V450 (Cat#560520, clone A20), anti-mouse CD45.2-V450 (Cat#560697, clone 104) anti-human IgM-APC (Cat#551062, clone G20-127), anti-human CD20-FITC (Cat#555622, clone 2H7), and anti-human CD19-APC (Cat#555415, clone HIB19) (all from BD Pharmigen). For proliferation analysis, cells were fixed and permeabilized using the Intracellular Fixation and Permeabilization Buffer Set (eBiosience) and stained with anti-ki67 (Abcam; Cat#ab16667); staining was revealed with alexa fluor 647 goat anti-rabbit IgG (H + L) (L.T.) (ThermoFisher, A-21245). Live cells were detected with DAPI (Sigma-Aldrich) or with LIVE/DEAD Fixable Yellow Dead Cell Stain (Thermo Fisher, L34959). Labeled cells were acquired with a LSRFortessa High-Parameter Flow Cytometer and analyzed with FlowJo V10.4.2 software.

Embryos and larvae were fixed in 4% PFA for 2 h at RT. After fixation, the embryos were sectioned by embedding them in 1.5% agarose/30% sucrose and frozen in 2-methylbutane chilled by immersion in liquid nitrogen. We collected 20 μm transverse sections on microscope slides using a cryostat microtome. Sections were rehydrated in PBS for 1 h and blocked with 5% goat serum/PBS for 1 h at RT. Primary antibody incubation was done overnight at 4°C. Secondary antibody incubation was done for 2 h at RT. Antibodies used were: rabbit anti-Sox10 (1:5000) (Binari et al. 2013 (link)), mouse 3D4 anti-Met (1:100; ThermoFisher), Alexa Fluor 647 goat anti-rabbit IgG(H+L) (1:1000), and Alexa Fluor 568 goat anti-mouse IgG(H+L) (1:1000). Sections were covered with Aqua-Poly/Mount (Polysciences). A 63× oil objective (NA = 1.4) mounted on a motorized Zeiss AxioObserver Z1 microscope equipped with a Quorum WaveFX-X1(Quorum Technologies) or Andor CSU-W1 (Andor Oxford Instruments) spinning disc confocal system was used to capture all images. Images were imported into Image J and Adobe Photoshop and Illustrator. Adjustments were limited to levels, contrast, and cropping.