Digital sight camera

Cryo-embedded mouse brains were cut into 15-μm slices using a cryostat (Leica Biosystems, Wetzlar, Germany). For immunofluorescence staining, cryosections were fixed with 4% paraformaldehyde (PFA, Sigma-Aldrich Merck, Darmstadt, Germany) in phosphate buffered saline (PBS, Sigma) for 15 min at room temperature (RT), rinsed 2 × 10 min with PBS and stained with the following primary antibodies: rat monoclonal anti-mouse glycoprotein Ib (GPIb) (1:100; emfret ANALYTICS, Eibelstadt, Germany), and rat monoclonal anti-mouse CD31 (MCA2388GA, 1:100; Bio-Rad Laboratories, Hercules, CA, USA). As secondary antibodies, Cy2 anti-rat (122-225-167, 1:100; Dianova, Hamburg, Germany) and Cy3 anti-rat (712-165-150, 1:100; Dianova) were used. Images of the immunofluorescence staining were acquired using a Nikon Eclipse 50i microscope equipped with Digital Sight camera (Nikon, Tokyo, Japan) and processed further with NIS-Elements imaging program (Nikon). GPIb-positive platelet aggregates in CD31-positive intraparenchymal microvessels were counted on five microscopic fields of the perilesional frontoparietal cortex in two brain slices per animal in a blinded fashion.

Immunostaining of formalin-fixed, paraffin-embedded tumor xenograft sections was carried out essentially as described [26 (link), 27 (link), 53 (link)]. Images were acquired by a Nikon ECLIPSE microscope and Digital Sight Camera (Nikon, NY, USA).

Internal reproductive organs were dissected from newly eclosed males in PBS, placed in a drop of PBS on a coverslip and punctured. A glass microscope slide was placed on top of the coverslip to squash the testes, and phase-contrast images were collected within 30 min of dissection using an Olympus IX-71 inverted microscope with Nikon Digital Sight camera with DS-U2 controller (20x lens/0.40 NA).

Extracellular RH or FRM2/3-KO parasites were placed on gelatin-coated glass and stimulated with BIPPO (5 μM). The type of movement of 100 parasites was scored in three independent experiments. Images were taken by Nikon digital sight camera at 25 frames per second on a Nikon eclipse Ti inverted microscope using a 63 x oil immersion objective.

Whole adult eyes were photographed with the Nikon Digital Sight camera mounted on a Nikon Eclipse 90i microscope equipped with a 12V, 100W halogen lamp by using a Nikon CFI60 (Chromatic Aberration Free Infinity) Plan Fluor 10x objective with numerical aperture 0.30. Z-stacks of adult eye images were flattened by using the NIS-Elements Imaging Software. Digital images were assembled using the Adobe Photoshop software. No biased image manipulations were applied.

Following euthanasia, intestinal segments were collected, flushed with cold PBS, and fixed in 10% neutral buffered formalin. Intestinal segments were removed from the fixative and sliced into five 10 mm sections, and placed into a tissue cassette. Tissue samples were dehydrated through a graded alcohol series, cleared with xylene, and embedded in paraffin wax. Tissue samples were then sliced into 5 µm sections using a microtome, mounted onto slides (5 sections per slide), and stained with hematoxylin and eosin stain for determination of gross morphology of intestinal villus height (VH) and crypt depth (CD). Images of intestinal sections were taken using an OLYMPUS^® BX50 microscope (Evident Corporation, Tokyo, Japan) equipped with a Nikon Digital Sight camera. Five cross-sections per slide per animal were viewed, and a total of 12 VHs and 12 CDs were photographed for each animal, and analyzed using NIS-Elements AR 3.10 software (Nikon Instruments Inc., Melville, NY, USA).