Image processing was completed in Zeiss Zen 2010, Adobe Photoshop CS4, and ImageJ (line scan). Quantification of sporozoite numbers and SG architecture features was done in Microsoft Excel. Statistical analyses and graphing were completed using Minitab 17 and Microsoft Excel. The original image stack capture files are available upon request. Raw data and analyses of infected SG quantification are provided in Data Set S1 .
Zen 2010
Manufactured by Adobe
The Zen 2010 is a versatile lab equipment product. It is designed to perform various analytical and experimental tasks in a laboratory setting. The core function of the Zen 2010 is to provide accurate and consistent measurements and data collection.
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Lab products found in correlation
710 confocal microscope, by Zeiss (4 mentions)
Imagej, by Adobe (3 mentions)
Alexa 594, by Thermo Fisher Scientific (2 mentions)
Alexa 488, by Thermo Fisher Scientific (2 mentions)
Alexa488 phallodin, by Thermo Fisher Scientific (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Plan apochromat 63x 1.4 na oil immersion objective, by Zeiss (1 mentions)
Paraformaldehyde (pfa), by Polysciences (1 mentions)
Rabbit anti ph3 s10 and s28, by Cell Signaling Technology (1 mentions)
Vectashield containing dapi, by Vector Laboratories (1 mentions)
6 protocols using zen 2010
1
Quantitative Analysis of Sporozoite Infection
2
Lysosomal Cargo Trafficking Regulation
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Cells were incubated with 25 µg/ml DQ-BSA Green in media at 37°C overnight, and chased for 2 h in normal culture media. For P/Q-type VGCC blocker treatment, ω-agatonxin TK at different concentrations (50 nM, 100 nM, 250 nM, 500 nM, and 1 µM) or Bepridil hydrochloride (10 µM) were added to the culture together with DQ-BSA. The following steps were the same as described above. Individual coverslips were washed twice with PBS, and fixed (4% PFA, 15 min) cells were then stained with anti-Lamp1 and DAPI.
All the mounted samples were examined and imaged on a confocal microscope LSM710 (Carl Zeiss) outfitted with a Plan‐Apochromat 63X 1.4NA oil immersion objective (Carl Zeiss). Data were collected using Carl Zeiss software ZEN 2010 and processed in Image J and Photoshop CS (Adobe).
All the mounted samples were examined and imaged on a confocal microscope LSM710 (Carl Zeiss) outfitted with a Plan‐Apochromat 63X 1.4NA oil immersion objective (Carl Zeiss). Data were collected using Carl Zeiss software ZEN 2010 and processed in Image J and Photoshop CS (Adobe).
3
Immunofluorescence Staining of Tissues
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Immunofluorescence: Tissues were dissected in PBS and fixed for 30–40 min in 4% para-formaldehyde (Polysciences, Inc.). After fixation samples were washed 3 times in PBS + 0.1% Triton X-100 (PBST) and incubated in primary antibodies overnight at 4°C. Samples were then washed as described and subjected to secondary antibody staining for 2 hr at room temperature followed by washing and mounting on Vectashield containing DAPI (Vector Laboratories, Inc.). Primary and secondary antibodies were incubated in PBST+ 0.5% BSA. Rabbit anti-pH3 S10 and S28 1:100 (Cell Signaling); rabbit anti-Bursicon (α-subunit) 1:250 (Luan H, Lemon WC, Peabody NC, 2006). Anti-mouse or anti-rabbit secondary antibodies Alexa 488 or Alexa 594 (Invitrogen) were used at 1:200 or 1:100, respectively. F-Actin was visualized with Alexa 488-Phallodin (Invitrogen) 1:500. Nuclei were counterstained with DAPI. Confocal images were collected using a Zeiss 710 Confocal microscope and processed with Zeiss ZEN 2010, ImageJ or Adobe Photoshop CS.
4
Confocal Microscopy Protocol for Gland Imaging
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Imaging was conducted using either a Zeiss LSM700 or LSM780 laser scanning confocal microscope housed in the Johns Hopkins University School of Medicine Microscope Facility. The step size used in 3D image stack captures was one micron. Each figure is composed of representative images from between six and 20 imaged glands, selected from 30 to 200 dissected and stained glands per experiment, processed between one and four different days. Image processing was completed in Zeiss Zen 2010 and Adobe Photoshop CS4.
5
Confocal Microscopy Protocol for Gland Imaging
6
Fluorescent Microscopy Imaging of Cytoskeleton
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The secondary antibodies used were as follows: goat anti-mouse, goat anti-chicken, or goat anti-rabbit Alexa 488, 1:200 (Invitrogen); and goat anti-mouse or goat anti-rabbit Alexa 594, 1:100 (Invitrogen). F-Actin was visualized with Alexa 488-Phallodin 1:500 (Invitrogen). Nuclei were counterstained with DAPI. Confocal images were collected using a Zeiss 710 Confocal microscope and processed with Zeiss ZEN 2010, ImageJ, or Adobe Photoshop CS.
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