Screening and imaging of HlpA-GFP S. pneumoniae-infected and non-infected zebrafish embryos were performed with a Leica MZ16FA fluorescence microscope with a Leica DFC420C camera attached. Non-infected control zebrafish embryos and embryos with visible fluorescent bacteria after infection were selected at previously determined time points and fixated overnight in 4 % paraformaldehyde in PBS. Subsequently, the embryos were stored in 100 % methanol at −20 °C for maximal of 2 month or until further use. Confocal images were generated with a Leica TCS SP8 Confocal Microscope. For optimal imaging, embryos were embedded in 1.5 % low-melting-point agarose dissolved in PBS in an open uncoated 8-well microscopy μ-Slide (http://ibidi.com ). Leica Application Suite X software was used to process the confocal images, specifically for brightness/contrast enhancements as well as for creating merged images.
Mz16fa fluorescence microscope
Manufactured by Leica
Sourced in Germany, Switzerland
The Leica MZ16FA is a fluorescence microscope designed for versatile imaging applications. It features high-performance optics, a wide range of magnification, and advanced illumination systems to enable high-quality imaging of fluorescent samples.
Automatically generated - may contain errors
Lab products found in correlation
Dfc420c camera, by Leica (4 mentions)
Application suite x software, by Leica (2 mentions)
Tcs sp8 confocal microscope, by Leica (2 mentions)
Dfc420c, by Leica (1 mentions)
Ms 222, by Merck Group (1 mentions)
Sp8 confocal microscope, by Leica (1 mentions)
Tricaine, by Merck Group (1 mentions)
Tcs sp8 x confocal microscope, by Leica (1 mentions)
Eight well microscopy μ slide, by Ibidi (1 mentions)
8 protocols using mz16fa fluorescence microscope
1
Fluorescent Imaging of S. pneumoniae Infection in Zebrafish
2
Generation of Stable Transgenic Zebrafish
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At 4 dpf, larvae injected with the construct were analysed for transgenic expression with a Leica MZ16FA fluorescence microscope. F0-embryos expressing EGFP in the brain region were selected and grown until reproducing age. Subsequent selection took place and F1 larvae with good expression were used for egg production. F2 larvae were used for further analysis and experiments described here. Stable germline transgenics TgBAC(cldn5a:eGFP)vum1 and Tg(kdrl:mCherry)is5;TgBAC(cldn5a:eGFP)vum2 were generated and used for the experiments.
3
Determining MTD of [2](PF6)2 in Zebrafish
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For determining the MTD of the water administration (WA) of the [2](PF6)2 solution in wild type zebrafish, solutions of 0.1 μM, 0.25 μM, 0.5 μM, 1 μM, 2 μM were made before the experiment. At 2.5, 3.5, 4.5, 5.5 dpf, [2](PF6)2 was added to the fish water and maintained for 12 h. At 3, 4, 5, 6 dpf, the fish water was refreshed and after 1 h, embryos were exposed to green light for 90 min (21 mW cm−2, 114 J cm−2, 520 nm). For the IV and RO administration, [2](PF6)2 solution (50 μM, 100 μM, 200 μM, 300 μM, 500 μM) was made before the experiment. At 3, 4, 5, 6 dpf, 1 nL of [2](PF6)2 was injected via the dorsal vein or the RO site and maintained for 1 h. Embryos were exposed to green light for 90 min (21 mW cm−2, 114 J cm−2, 520 nm). The images of treated and wild type embryos at 6 dpf were taken using a DFC420C camera coupled to a Leica MZ16FA fluorescence microscope. In order to determine the MTD of tumour cell-bearing zebrafish, 90 min green light activation (21 mW cm−2, 114 J cm−2, 520 nm) was performed according to the same procedure, after [2](PF6)2 was delivered by WA, IV and RA administration as described above for the wild type embryos.
4
Quantifying Metastatic Capacity in Zebrafish Xenografts
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For each drug treatment experiment 20 injected zebrafish larvae were randomly selected and imaged using a MZ16FA fluorescence microscope equipped with a DFC420C camera (Leica, Wetzlar, Germany) as described [49 ]. Representative engrafted phenotypes were subjected to confocal imaging; to this end zebrafish larvae were anaesthetized with 0.002% tricaine (MS222, Sigma) in zebrafish medium. After embedding the larvae in 1% low melting temperature agarose/zebrafish medium, images were recorded for both green (GFP) and red (tdTomato/CMDiI) channels. Whole larva stitches were generated at 10x magnification using a Leica sp8 confocal microscope (Leica, Wetzlar, Germany). Consecutive stitch sequences were processed into a single image using Fiji 30 using a previously described plugin [53 (link)]. Metastatic capacity or tumor burden at the end of the experiment was determined as the normalized fluorescence intensity of the engrafted cells (either normalized to 1 dpi for time course experiments or to vehicle treated control for treatment experiments).
5
Antimycobacterial Effect of Antibiotics in Zebrafish
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To assess the antimycobacterial effect of Q203, clofazimine and BDQ, zebrafish embryos were infected with 100 colony forming units (CFUs) in the caudal vein by microinjection with M. marinum MUSA containing the plasmid pSMT3-hsp60-mEos3.1. (Stoop et al./van Leeuwen et al. unpublished). The embryos were treated 1 day post infection with the aforementioned antibiotics at the indicated concentrations. Fluorescence was monitored at specific time-points with a Leica MZ16FA fluorescence microscope and a Leica DFC420C camera. All experiments were performed at least three times. For expression analysis in zebrafish embryos pMV-hsp60-mEos3.168 (link) was introduced in M. marinum MUSA already containing the cydA reporter plasmid. Zebrafish embryos were injected with 2000 CFUs by microinjection in the hindbrain ventricle, as previously described24 (link), 41 (link). Subsequently, zebrafish embryos were fixated O/N at 0, 2 and 4 days post infection in 4% paraformaldehyde dissolved in PBS, washed with PBS and subsequently stored in PBS at 4 °C. For confocal microscopy, zebrafish were mounted in 1% low-melting-point agarose in PBS and imaged with a Leica TCS SP8 confocal microscope.
6
Mycobacterium marinum Infection in Zebrafish
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At 2 or 4 dpf, embryos were infected with M. marinum 2012 ). At 1–5 dpi bacterial infection was monitored daily with a Leica MZ16FA fluorescence microscope. Following analysis, larvae were fixated on indicated time points overnight in 4% (v/v) paraformaldehyde (EMS, 100122) in PBS, and stored in 100% methanol at −20 °C for immune‐histochemical staining and confocal imaging. To determine the exact number of bacteria injected, the injection volume was plated on 7H10 plates containing the proper antibiotic selection. During injection and microscopic examining, larvae were anaesthetised in egg water with 0.02% (W/V) buffered 3‐aminobenzoic acid (Tricaine; Sigma‐Aldrich, A‐5040).
7
Visualizing Early Granuloma Formation
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Bacterial infection was monitored initially with a Leica MZ16FA fluorescence microscope. Bright field and fluorescence images were generated with a Leica DFC420C camera. Early granuloma formation was analysed visually and quantified with custom‐made pixel‐counting software (http://www.elaborant.com ). Confocal analysis was performed on hCMEC/D3 cells and larvae, embedded in 1% low melting‐point agarose (Boehringer Mannheim, 12841221‐01) in an eight‐well microscopy μ‐slide (ibidi). Analysis was performed with a confocal laser‐scanning microscope (Leica TCS SP8 X Confocal Microscope). Leica Application Suite X software and ImageJ software were used to adjust brightness and contrast and to create overlay images and 3D models.
8
Green Light Exposure of Zebrafish Embryos
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Two dpf embryos were transferred into 6-well plates (10 embryos/well). The embryos were exposed to green light (21 mW/cm2, 520 nm) for 0, 3, 6, 12 h. After irradiation, images were taken using a DFC420C camera coupled to a MZ16FA fluorescence microscope (Leica, Heerbrugg, Switzerland).
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