Grca er camera

Immunofluorescence assays (IFA) on cells fix with 4% formaldehyde (Polysciences, Warrington, PA) plus 0.02% glutaraldehyde in PBS and permeabilize with 0.3% TritonX-100 was performed as described previously [24 (link)]. Coverslips were mounted using ProLong Diamond Antifade Mountant (Life Technologies) to minimize bleaching during microscopy observations. Cells were viewed with a Nikon Eclipse 90i equipped with an oil-immersion plan Apo 100x NA 1.4 objective and a Hamamatsu GRCA-ER camera (Hamamatsu Photonics, Hamamatsu, Japan). Optical z-sections with 0.2-μm spacing were acquired using Volocity software (PerkinElmer, Waltham, MA). The images were deconvolved using an iterative restoration algorithm and the registry was corrected using Volocity software. The positive product of the differences of the mean (PDM) images were calculated using Volocity software, which was also used to adjust brightness levels, cropping and resizing of the images obtained. Images of PV shown are representative of at least 20 PV.

Confluent Hepa1-6 cells seeded in a 24-well plate were infected with 5000 freshly dissected sporozoites. After centrifugation of the plate at 400×g for 5 min for sporozoites contacting mammalian cells, monolayers were incubated at 37 °C for 3 h before washing with PBS to remove extracellular (noninvading) sporozoites. Infected cells were washed, fixed, and immunostained at different time points as described^{18 (link),65 (link)}. Fixed samples were viewed with either a Nikon Eclipse 90i fluorescence microscope equipped with an oil-immersion Nikon plan Apo ×100/NA 1.4 objective, or a Nikon plan Fluor ×60/NA 0.75 objective and a Hamamatsu GRCA-ER camera (Hamamatsu Photonics, Hamamatsu, Japan) or a Zeiss AxioImager M2 fluorescence microscope equipped with an oil-immersion Zeiss plan Apo ×100/NA 1.4 objective and a Hamamatsu ORCA-R2 camera. Optical z-sections with 0.2 µm spacing were acquired using the Volocity 6.3.1 software acquisition module (Perkin Elmer, Waltham, MA).