Super coolscan 9000 scanner

IHC were performed on 5-μm tumor sections in the Department of Pathology of the Centre Hospitalier Universitaire de Sherbrooke, Québec (CHUS) (Sherbrooke) using a standard streptavidin-biotin-peroxidase immunostaining procedure with a Ventana NexES autostainer and the solvent-resistant DAB Map detection kit (Ventana Medical Systems, Tucson, AZ) using ready-to-use solutions (Ki67 and E-cadherin) purchased from Dako, Burlington, Ontario, Canada.
Ki67-positive cells were manually counted in up to five × 400 light microscope representative fields per tumor (containing an average of 150 cells). Total counts were reported as total cell number per field. E-cadherin protein levels were quantified using the yellow channel of a cyan, magenta, yellow, key (CMYK) color model with pictures taken with a Super Coolscan 9000 scanner (Nikon, Tokyo, Japan) using Fiji software (Open Source) [13] (link), and quantification was performed using Image-Pro software (Media Cybernetics, Bethesda, MD). To avoid quantification of any nontumoral area (e.g., skin and fat), the xenograft sections were counterstained using hematoxylin and eosin in addition to staining the estrogen receptor, a positive marker of SKOV3 cells. Pictures with × 100 and × 400 magnifications were acquired using an Axioskop 2 phase-contrast microscope (Carl Zeiss, Thornwood, NY) and processed using Image-Pro software.

To vitrify particles for cryo-EM observation from defined conditions of elevated temperature and humidity, we used a custom-made environmental chamber that mounts over a cryo-station (see Fig. S1 in the supplemental material). In brief, 3-μl drops of specimen were applied to EM grids mounted within the chamber, incubated for 10 min at 65°C or 70°C, and then blotted to thin films and vitrified in an otherwise conventional manner. Finally, grids were transferred into the electron microscope and low-dose micrographs were recorded on a CM200-FEG electron microscope (FEI, Hillsboro, OR) equipped with a Gatan 626 cryo-holder using procedures previously described (43 (link)). The Head I* sample was imaged at a magnification of ×38,000 and defocus values in the range of 0.78 µm to 2.15 µm. Films were digitized with a Zeiss SCAI scanner with a 7-µm sampling rate to yield 1.84 Å/pixel at the sample. The Head I sample was imaged at a magnification of ×50,000, and films were scanned on a Nikon Super CoolScan 9000 scanner with a 6.35-µm sampling rate, yielding 1.27 Å/pixel. Capsid diameters were measured by hand as averages of three measurements at positions 60° apart for each capsid. To estimate the incidence of damaged capsids, ~400 capsids from three representative micrographs were scored independently for each sample by two observers.