Volocity 3d image analysis software

The htert-RPE-1 cells were fixed in 4% formaldehyde (Thermo Fisher Scientific) in PBS for 20 min at room temperature, treated with 0.25% NH₄Cl, and permeabilized by 0.1% Triton X-100 in PBS for 10 min. The cells were then blocked with 5% NDS (Jackson ImmunoResearch) in PBS for 30 min, followed by primary antibody staining for 1 h and secondary antibody staining for 45 min at room temperature. Primary antibodies used in this study were sheep anti-NOTCH3 ECD (1:500; R&D systems), rabbit anti-NOTCH3 ICD (D11B8, 1:500; Cell Signaling Technology), mouse anti-EEA1 (E9Q6G, 1:200; Cell Signaling Technology), and rabbit anti-clathrin heavy chain (Cat# 4796, RRID:AB_10828486; Cell Signaling Technology, used 1:50).
Images were captured using Volocity (Volocity 3D Image Analysis Software, RRID:SCR_002668) with an Orca-ER digital camera (Hamamatsu) mounted on a M2 fluorescent microscope (Zeiss). Deconvolution was performed with three nearest neighbors using Openlab (Improvision) and processed in Photoshop (RRID:SCR_014199; Adobe Photoshop).

For imaging sporozoite invasion and confocal imaging of developing exoerythrocytic forms, cells were visualized using a Nikon 90i microscope at 100× magnification, and images were acquired with a Hamamatsu Orca-ER camera using the Volocity 3D Image Analysis Software with image stacks deconvolved prior to combining and focused along the plane of the parasites. Epifluorescence imaging of exoerythrocytic forms was performed using an Olympus BX-53 upright microscope at 100× magnification. Imaging of hepatocytes following protein staining was performed using either a Zeiss Axioskop 2 microscope with a ProgRes MFcool camera using the ProgRes CapturePro software version 2.10.0.1 or using a BZ-X710 (Keyence, Osaka, Japan) All-in-One Fluorescence Microscope. BZ-X700 Analyzer Software (version 1.31.1) was used for Z-stack analyses to yield a single compressed fully focused image at 600× magnification from 9 to 11 planes, with each plane at 0.2 μm thickness.