All slides were imaged under 10× magnification using a Carl Zeiss AxioObserver Z1 (Zeiss, Inc.) Fluorescently labeled tissue was imaged utilizing an AxioCam MRm monochrome camera (Zeiss, Inc.). DAB labeled tissue was imaged using an AxioCam ERc5 color camera (Zeiss, Inc.). In order to capture the entire area of implantation, the Mosaix module was used to stitch together a 4 × 4 tile image. Images shown have been enhanced to improve visual representation.
Axiocam mrm monochrome camera
Manufactured by Zeiss
Sourced in Germany
The AxioCam MRm is a monochrome camera designed for microscopy applications. It features a high-resolution sensor that captures detailed images. The camera supports various image acquisition modes and interfaces for integration into microscopy systems.
Automatically generated - may contain errors
Lab products found in correlation
Axio zoom v16, by Zeiss (10 mentions)
Axioskop2 mot plus microscope, by Zeiss (3 mentions)
Carl axio observer z1, by Zeiss (1 mentions)
Axiovision software, by Zeiss (1 mentions)
Volocity software, by PerkinElmer (1 mentions)
Alexa fluor 594 conjugated goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated goat anti mouse igg secondary antibody, by Thermo Fisher Scientific (1 mentions)
Argon laser, by Zeiss (1 mentions)
Plan apochromat 63 1.4 oil dic m27 objective, by Zeiss (1 mentions)
Zen 2012, by Zeiss (1 mentions)
Axioskop 2 plus fluorescence microscope, by Zeiss (1 mentions)
Planapo z 0.5 objective, by Zeiss (1 mentions)
Zen pro, by Zeiss (1 mentions)
Axio zoom v16 microscope, by Zeiss (1 mentions)
Hank s balanced salt solution, by Merck Group (1 mentions)
Cl 9000, by Zeiss (1 mentions)
11 protocols using axiocam mrm monochrome camera
1
Mosaic Imaging of Fluorescent and DAB Tissues
2
Studying Parasite Invasion Dynamics
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After harvesting by mechanical lysis, parasites were transferred to intracellular buffer (5 mM NaCl, 142 mM KCl, 1 mM MgCl2, 2 mM EGTA, 5.6 mM d -glucose, 25 mM HEPES, pH 7.4) and allowed to invade subconfluent HFF cell monolayers on coverslips in 24-well plates for 90 s. The 24-well plates were then immediately transferred to an ice water bath. Coverslips were rinsed, fixed with 4% formaldehyde, and processed for immunofluorescence. To mark extracellular parasites, cells were stained with MAb DG52 prior to permeabilization with 0.05% saponin. After permeabilization, anti-GFP antibody was used to mark Δact1 parasites, a rabbit polyclonal antibody to RON4 provided by John Boothroyd was used to mark the moving junction, and fluorophore-conjugated secondary antibodies (for DG52, Alexa Fluor 350; for GFP antibody, Alexa Fluor 488; and for RON4 antibody, Alexa Fluor 594) were used for visualization. Images were acquired on a Zeiss Axioskop 2MOT plus microscope, using an AxioCam MRm monochrome camera and AxioVision software (Carl Zeiss, Inc., Thornwood, NY).
3
Quantifying Parasite Invasion Dynamics
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Freshly egressed parasites of the WT and Δrom1 Δrom4 Δrom5 strains were treated with 3% FBS plus 1% ethanol to induce their microneme secretion for 15 min in invasion medium. Subsequently, they were allowed to invade HFF monolayers for 5 min, fixed with 4% paraformaldehyde, and subjected to immunostaining. Samples were first stained with rabbit anti-SAG1 followed by Alexa 594-conjugated goat anti-rabbit secondary antibody. Following 0.1% saponin permeabilization, samples were stained with MAb 6D10 (for MIC2), MAb B3.90 (for AMA1), or MAb DG52 (for SAG1) and visualized by Alexa 488-conjugated goat anti-mouse secondary antibody. Parasite and host nuclei were stained by 4′,6-diamidino-2-phenylindole (DAPI). Images were acquired using a Zeiss Axioskop 2 MOT Plus microscope equipped with a 63× numerical-aperture (NA)-1.6 oil immersion lens and an AxioCam MRm Monochrome camera (Carl Zeiss, Inc., Thornwood, NY). Fluorescent signals were quantified by the Volocity software (PerkinElmer, Waltham, MA). Two independent experiments (each with triplicates) were conducted.
4
CRISPR/Cas9 Gene Disruption Frequency
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To estimate the frequency of CRISPR/CAS9-mediated gene disruptions, RH parasites (~107) were transfected with 7.5 µg of indicated CRISPR plasmids (Table S1 ) and analyzed by immunofluorescence staining for GFP 24 h posttransfection. HFF monolayers were fixed with 4% formaldehyde, permeabilized with 0.1% Triton X-100, and incubated with MAb 3E6 (Life Technologies, Carlsbad, CA) to detect GFP. Parasites were detected with rabbit anti-TgALD (38 (link)). Alexa Fluor 594-conjugated goat anti-rabbit IgG and Alexa Fluor 488-conjugated goat anti-mouse IgG secondary antibodies (Life Technologies, Carlsbad, CA) were used to detect primary antibodies. Images were acquired using a Zeiss Axioskop 2 MOT Plus microscope equipped with a 63×, NA (numerical aperture) 1.6 oil immersion lens and an AxioCam MRm monochrome camera (Carl Zeiss, Inc., Thornwood, NJ).
5
Fluorescent Imaging of Pellicles
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Fluorescent images of pellicles were obtained with an Axio Zoom V16 stereomicroscope (Carl Zeiss, Jena, Germany) equipped with a Zeiss CL 9000 LED light source and an AxioCam MRm monochrome camera (Carl Zeiss) and HE eGFP (excitation at 470/40 nm and emission at 525/50 nm), and HE mRFP (excitation at 572/25 nm and emission at 629/62 nm) filter sets. Images were taken at 3.5× and 55× magnifications. The exposure times for green and red fluorescence were set up to maximal possible values before reaching overexposure, using range indicator function. Zeiss software was used to obtain overlaid, artificially colored images of both fluorescence channels.
6
Automated Microscopy for Pellicle Imaging
7
Resveratrol Modulation of NTHi Infection
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A549 cells were seeded on 13 mm circular coverslips in 24-well tissue culture plates and NTHi infected for 2 h as previously described15 (link). Infected cells were incubated in RPMI 1640 containing 10% FCS, Hepes 10 mM and gentamicin 200 μg/ml for 1 h. When necessary, cells were pre-treated with resveratrol 3 μM or DMSO for 4 h in 1 ml EBSS, maintained during bacterium-cell contact; alternatively, cells were infected, and resveratrol 20 μM or DMSO was added during the gentamicin incubation period. Cells were washed three times with PBS and fixed with 3.7% paraformaldehyde (PFA) in PBS pH 7.4 for 15 min at room temperature. Bacteria were stained with rabbit anti-NTHi serum15 (link) diluted 1:600. Late endosomes were stained with mouse monoclonal anti-human Lamp-1 H4A3 antibody (Developmental Studies Hybridoma Bank) diluted 1:70. Donkey anti-rabbit conjugated to Alexa 488 and donkey anti-mouse conjugated to Rhodamine Red-X (RRX) secondary antibodies (Jackson Immunological) were diluted 1:100. Samples were analyzed with a Carl Zeiss Axioskop 2 plus fluorescence microscope and a Carl Zeiss Axio Cam MRm monochrome camera.
8
Pellicle Formation Assay for Barcoded Bacterial Strains
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We examined the ability of all barcoded strains to form pellicles on MSgg medium. Overnight LB liquid cultures of all barcoded strains were adjusted to O.D.600 0.1. 20 µl of the O.D.-adjusted cultures was added to 2 ml of MSgg liquid medium in 2 ml microplate wells. These cultures were incubated at 30 °C for 5 days. After 2 days of incubation and at the end of the incubation period the obtained pellicles were examined with an Axio Zoom V16 stereomicroscope (Carl Zeiss, Germany) equipped with a Zeiss CL 9000 LED light source, a PlanApo Z ×0.5 objective, and AxioCam MRm monochrome camera (Carl Zeiss, Germany).
9
Visualizing Bacterial Pellicle and Root Colonization
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Fluorescent images of colonies and whole pellicle were obtained with an Axio Zoom V16 stereomicroscope (Carl Zeiss, Jena, Germany) equipped with a Zeiss CL 9000 LED light source and an AxioCam MRm monochrome camera (Carl Zeiss) and HE eGFP (excitation at 470/40 nm and emission at 525/50 nm), and HE mRFP (excitation at 572/25 nm and emission at 629/62 nm) filter sets. The exposure times for green and red fluorescence were set up to maximal possible values before reaching overexposure, using the range indicator function.
The pellicles formed in TSB medium at 24 h and root colonization at 24 h were visualized with a confocal laser scanning microscopy (CLSM, LMI-005-Leica Microsystems Confocal Microscope-SP8). Fluorescent reporter excitation was performed with the argon laser at 488 and 556 nm, the emitted fluorescence was recorded at 484−536 nm and 560−612 nm for GFP and DsRed, respectively. ImageJ software was used to obtain overlaid, artificially colored images for both stereomicroscope and CLSM.
The pellicles formed in TSB medium at 24 h and root colonization at 24 h were visualized with a confocal laser scanning microscopy (CLSM, LMI-005-Leica Microsystems Confocal Microscope-SP8). Fluorescent reporter excitation was performed with the argon laser at 488 and 556 nm, the emitted fluorescence was recorded at 484−536 nm and 560−612 nm for GFP and DsRed, respectively. ImageJ software was used to obtain overlaid, artificially colored images for both stereomicroscope and CLSM.
10
Visualizing Zebrafish Germline Cells
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Cells of the germline were identified by their expression of the vasa:vasa-eGFP transgene (Krøvel & Olsen 2002) (link). Embryos at 2 dpf were imaged directly, while larvae at 4 dpf, as well as fish at 2, 3, 6, 9 and 12 wpf were anesthetized as described previously before in vivo imaging. Because many zebrafish diets may cause nonspecific fluorescent signal within the digestive tract, all fish at 2 wpf and older were fasted for a minimum of 24 h before imaging. For gonad dissection, the zebrafish were killed as described previously and the gonad was removed under epifluorescent light using fine dissecting tools. For spermatozoa collection, mature males were anesthetized and patted dry with tissue paper before gently stroking the side of the fish. Spermatozoa were collected from the genital pore using a 10-µL pipette with gentle suction and diluted in 50 µL Hanks' balanced salt solution (Sigma Aldrich). All visualization of the gonads was performed using an AxioZoom V.16 microscope (Carl Zeiss) equipped with an AxioCam MRm monochrome camera (Carl Zeiss) and Zen Pro (2012; Carl Zeiss) imaging software. Images were taken using either bright field or epifluorescent light with an enhanced green fluorescent protein filter (excitation: 488 nm, emission: 509 nm).
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