Confocal imaging was performed using an LSM1 700 confocal microscope (Zeiss) equipped with 405-nm (5 mW fiber output), 488-nm (10 mW fiber output), 555-nm (10 mW fiber output) and 639-nm (5 mW fiber output) diode lasers, a main dichroic beam splitter URGB and a gradient secondary beam splitter for LSM 700, using a 10× EC Plan-Neofluar (10×/0.3) or a 20× Plan-Apochromat (20×/0.8) objective (Zeiss, Munich, Germany). Image acquisition was performed with ZEN 2010 (Zeiss), and image dimensions were 1024×1024 pixels with an image depth of 16 bit. Two times averaging was applied during image acquisition. Laser power and gain were adjusted to avoid saturation of single pixels. All images were taken using identical microscope settings based on the secondary antibody control staining.
Plan apochromat 20 0.8 objective
Manufactured by Zeiss
Sourced in Germany
The Plan-Apochromat 20×/0.8 objective is a high-performance optical lens designed for microscopy applications. It features a magnification of 20× and a numerical aperture of 0.8, providing excellent image quality and resolution. The objective is plan-apochromatic, meaning it is corrected for both chromatic and spherical aberrations, ensuring accurate and detailed image reproduction.
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Lab products found in correlation
Lsm 710, by Zeiss (10 mentions)
Axio observer z1, by Zeiss (6 mentions)
Fluar 10 0.5 objective, by Zeiss (4 mentions)
Lsm 700, by Zeiss (4 mentions)
Zen 2010, by Zeiss (3 mentions)
Hoechst 33342, by Thermo Fisher Scientific (3 mentions)
Lsm 780 confocal microscope, by Zeiss (3 mentions)
Lsm 700 confocal microscope, by Zeiss (2 mentions)
Airyscan detector, by Zeiss (2 mentions)
Flash4.0 v3 scmos camera, by Hamamatsu Photonics (2 mentions)
Axio observer z1 7 body, by Zeiss (2 mentions)
Axiocam 506 mono camera, by Zeiss (2 mentions)
Lsm 700 microscope, by Zeiss (2 mentions)
Axio observer z1 microscope, by Zeiss (2 mentions)
Zen blue software, by Zeiss (2 mentions)
Plan neofluar 40 1.3 oil objective, by Zeiss (1 mentions)
P phenylenediamine, by Merck Group (1 mentions)
Axio imager z1 microscope, by Zeiss (1 mentions)
Goat anti cd36, by R&D Systems (1 mentions)
Zen software, by Zeiss (1 mentions)
31 protocols using plan apochromat 20 0.8 objective
1
Confocal Imaging of Fluorescent Samples
2
Confocal Microscopy Imaging Protocol
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All images were taken on an LSM 700 confocal microscope (Zeiss) equipped with 405-nm (5-mW fiber output), 488-nm (10-mW fiber output), 555-nm (10-mW fiber output), and 639-nm (5-mW fiber output) diode lasers using a 20× Plan-Apochromat (20×/0.8) objective (Zeiss). Image acquisition was done with ZEN 2010 (Zeiss). Image dimensions were 1024 × 1024 pixels with an image depth of 12 bits. Two-times averaging was applied during image acquisition. Laser power and gain were adjusted to avoid saturation of single pixels. Adjustment of brightness/contrast and changing of artificial colors (LUT) were done in Fiji/ImageJ. The same imaging parameters and adjustments were used for all images within an experiment.
3
Fluorescence Imaging of Brain Sections
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Digital camera (HDC-HS900GK) was used to acquire the bright-field images of samples. Confocal fluorescence microscopy (LSM710, Zeiss, Germany), equipped with the Fluar objective (dry, working distance 2.0 mm) and Plan-Apochromat objective (dry, working distance 0.55 mm), was used to acquire the GFP fluorescence images of brain sections. Before and after clearing, the fluorescence images were obtained under the same imaging parameters.
4
Confocal and Widefield Imaging of Cryptococcus
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Confocal imaging was performed on a Zeiss Axio Observer.Z1/7 body equipped with a Zeiss Airyscan detector. All channels were collected using Airyscan Multiplex settings with default processing. Cryptococcal India ink and lectin staining images (Fig. 3 ; Fig. S3) were captured using a Zeiss LD C-Apochromat 60×/1.1 Oil Korr UV VIS IR objective. Correlative DIC images in Fig. S2 were captured shortly after the confocal collection in widefield mode using a Zeiss 40×/1.1 Water corrected Plan Apochromat objective and re-scaled manually to overlay with confocal images taken with the same objective. All other images were captured using a Zeiss Plan-Apochromat 20×/0.8 objective. Live imaging was performed with larvae anesthetized in tricaine as previously described (48 (link)) and simply resting on the bottom of a glass-bottom dish or immobilized in 1% low melt agarose. For the collection of large numbers of events, a combination of widefield and confocal imaging was used to create scout and detail images for later analysis. Widefield imaging was performed using the same Zeiss optical setup, with image capture using a Hamamatsu Flash4.0 V3 sCMOS camera. Widefield fluorescence excitation was generated with a Colibri 7 type RGB-UV fluorescence light source. The filter set was Zeiss set 90 LED.
5
Immunofluorescent Staining of Tissue Sections
6
Plaque and Intracellular Growth Assays for Toxoplasma
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Plaque assays: HFF monolayers were infected with parasites and let to develop for 7 days before fixation with PFA/GA and Giemsa staining (Sigma-Aldrich GS500) mounted with Fluoromount G and visualized using ZEISS MIRAX imaging system equipped with a Plan-Apochromat 20×/0.8 objective at the bioimaging facility of the Faculty of Medicine, University of Geneva.
Intracellular growth assays: Prior to infection, the HFF monolayers were washed and pre-incubated for 24 h with medium containing the relevant carbon source and kept in this medium for the rest of the experiment. Complete medium is DMEM 41966 (Gibco, Life Technologies) supplemented with 5% FCS, up to 6 mM glutamine, 25 µg/ml gentamicine. Medium depleted in glutamine is DMEM 11960 supplemented with 25 µg/ml gentamicine (Gibco, Life Technologies) and medium depleted in glucose is DMEM 11966 supplemented with up to 6 mM glutamine and 25 µg/ml gentamicine. HFF were inoculated with parasites and coverslips were fixed 24 h post-infection with 4% PFA and stained by IFA with rabbit anti-TgGAP45, mouse anti-myc 9E10. Number of parasites per vacuole was counted in triplicates for each condition (n = 3). More than 200 vacuoles were counted per replicate.
Intracellular growth assays: Prior to infection, the HFF monolayers were washed and pre-incubated for 24 h with medium containing the relevant carbon source and kept in this medium for the rest of the experiment. Complete medium is DMEM 41966 (Gibco, Life Technologies) supplemented with 5% FCS, up to 6 mM glutamine, 25 µg/ml gentamicine. Medium depleted in glutamine is DMEM 11960 supplemented with 25 µg/ml gentamicine (Gibco, Life Technologies) and medium depleted in glucose is DMEM 11966 supplemented with up to 6 mM glutamine and 25 µg/ml gentamicine. HFF were inoculated with parasites and coverslips were fixed 24 h post-infection with 4% PFA and stained by IFA with rabbit anti-TgGAP45, mouse anti-myc 9E10. Number of parasites per vacuole was counted in triplicates for each condition (n = 3). More than 200 vacuoles were counted per replicate.
7
HEK293T-CD63-eGFP Microscopic Imaging
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1–2 µL of the obtained HEK293T-CD63-eGFP samples were transferred to microscopic slides and analysed by an Axio Observer.D1 fluorescent microscope with a Plan-Apochromat 20×/0.8 objective (Zeiss).
8
Tissue Staining and Imaging Protocol
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Tissues were processed, sectioned and stained as previously described65 (link). PicroSirius Red staining was performed as recommended by the manufacturer (ab150681, Abcam, Cambridge, United Kingdom). Colorimetric images were captured using Axio Scan.Z1 with Plan Apochromat 20×/0.8 objective (Carl Zeiss, Germany). Fluorescence images were captured using the LSM 710 confocal microscope with the Zeiss EC Plan-NEOFLUAR 20×/0.5 NA objective. Analyses were performed with ZEN 2012 Light Edition software (Carl Zeiss, Germany).
9
Immunohistochemistry and Immunofluorescence of Microglia
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IHC and IF were performed as previously described (31 (link)). In brief, mice were deeply anesthetized and transcardially perfused with PBS and 4% paraformaldehyde (PFA). Brains were excised, postfixed in 4% PFA overnight, saturated in 30% sucrose solution, and sectioned with a cryotome at 30 μm. IHC staining of microglia was performed using anti-IBA1 antibody (Wako Chemicals, Neuss, Germany) and imaged using a Zeiss AXIO Scope A1 equipped with a Plan-APOCHROMAT 20×/0.8 objective. Images were acquired at room temperature with the AxioRel Vision 4.8 software and quantified in ImageJ. For double IF goat anti-GFAP antibody (Sigma-Aldrich) and rabbit anti-MGL antibody (kind gift from Ken Mackie, Indiana University Bloomington) and anti-goat (Alexa Fluor 488-conjugated) and anti-rabbit (Alexa Fluor 594-conjugated) secondary antibodies were used. Sections were imaged by confocal laser scanning microscopy using a Leica TCS SP5 II equipped with a HCX PL APO 63 × 1.3 NA objective. Images were obtained at room temperature with Leica LAS AF software.
10
Live Confocal Imaging of Anesthetized Larvae
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Confocal imaging was performed on a Zeiss Axio Observer.Z1/7 body equipped with a Zeiss Airyscan detector. All channels were collected using Airyscan Multiplex settings with default processing. Images in Fig. 4A were captured using a Zeiss LD C-apochromat ×40/1.1 W Korr UV-visible-infrared (UV-VIS-IR) objective. All other images were captured using a Zeiss Plan-apochromat ×20/0.8 objective. Live imaging was performed with larvae anesthetized under tricaine as previously described (49 (link)) and simply resting on the bottom of a glass-bottom dish or immobilizing in 1% low-melt agarose. For collection of large numbers of events, a combination of widefield and confocal imaging was used to create scout and detail images for later analysis. Widefield imaging was performed using the same Zeiss optical setup, with image capture using a Hamamatsu Flash4.0 V3 sCMOS camera. Widefield fluorescence excitation was performed with a Colibri 7-type RGB-UV fluorescence light source. The filter set was Zeiss set 90 LED.
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