Muscle cross-sections from uvula of all the patients and controls were included in the quantification. Palatopharyngeus muscles were not included in the quantitative analysis, due to the small sample size and lack of control biopsies. In uvula, 4 to 5 random areas from each muscle cross-section were scanned at 20x magnification with a fluorescence microscope (Leica DM6000B, Leica Microsystems CMS GmbH, Wetzlar, Germany) equipped with a digital high-speed fluorescence charge-coupled device (CCD) camera (Leica DFC360 FX). The number of unstained, weakly stained, or disorderly stained fibres for the Abs directed against desmin and dystrophin were quantified manually on each photo (Photoshop CS5, version 12.0.4, San Jose, CA, USA). The investigators were blinded to the origin of the samples. Comparisons between the mean values of changes in muscle fibres between the two groups were made using the Mann-Whitney U test. For comparing more than two groups, planned one-way ANOVA (analysis of variance) with bootstrapping was used. Both statistical tests do not require an assumption of normality in the distribution of data. Values are presented as the mean ± standard deviation. Results were considered significant at a p-value of ≤0.05. All the tests were performed with SPSS (statistical package for social sciences) software (IBM SPSS 23, statistical software, Armok, NY: IBM corp., USA).
Dfc360 fx
Manufactured by Leica
Sourced in Germany, United Kingdom
The Leica DFC360 FX is a high-performance digital camera designed for microscopy applications. It features a full-frame CCD sensor with a resolution of 6.3 megapixels, providing high-quality image capture. The camera offers a wide dynamic range and advanced color reproduction capabilities, making it suitable for a variety of microscopy techniques.
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Lab products found in correlation
Dm6000b, by Leica (8 mentions)
Dm5500b, by Leica (6 mentions)
Dm5500b microscope, by Leica (6 mentions)
Hoechst 33342, by Thermo Fisher Scientific (5 mentions)
Dfc420c, by Leica (5 mentions)
Dmi4000b microscope, by Leica (4 mentions)
Dmi4000b, by Leica (4 mentions)
M205 fa, by Leica (4 mentions)
Dmi6000b, by Leica (4 mentions)
Tcs sp5 2, by Leica (3 mentions)
In situ cell death detection kit, by Roche (3 mentions)
Dm6000, by Leica (3 mentions)
Dm4000b microscope, by Leica (3 mentions)
Las af software, by Leica (3 mentions)
Tsa cy3 system, by PerkinElmer (2 mentions)
Las af, by Leica (2 mentions)
Dfc480, by Leica (2 mentions)
M205fa microscope, by Leica (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Dm4000b, by Leica (2 mentions)
89 protocols using dfc360 fx
1
Quantifying Muscle Fiber Abnormalities in Uvula
2
Microscopy and Flow Cytometry of Candida
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Fungal cells were grown in DMEM+10% FBS on glass coverslips in a 24 well microtiter plate for filipin (Sigma), calcofluor white (CFW) and Als3 immunostaining. Flow cytometry was used to quantify mannan and β-1,3-glucan exposure on the surface of stationary C. albicans cells after staining with concanavalin A and anti- β-1,3-glucan. Piercing and invasion rate were determined by differential staining. All staining procedures are described in Protocol S1 . Epifluorescence (Leica DM5500B, Leica DFC360 FX) was used to detect CFW and filipin (DAPI filter), Alexa Fluor 488 (FITC filter) and Alexa Fluor 647 (Cy5 filter). Micrographs were taken with a Leica Digital Camera DFC360 FX or a Zeiss AxiCam ICc3.
3
Comprehensive Histopathological and Immunofluorescence Analysis
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Histopathological examinations were performed after Mayer’s H&E staining. Immunofluorescence staining was performed on formalin-fixed paraffin-embedded tissue by using the Tyramide Signal Amplification (TSA) Cy3 system (Perkin&Elmer) according to the manufacturer’s protocol. The following primary antibodies were used: Anti-Villin (Santa Cruz), anti-β-Catenin (Cell Signaling), anti-Caspase-8 (Cell Signaling) and anti-Cleaved Caspase-8 (Cell Signaling). A biotinylated anti-rabbit secondary antibody from Dianova was used. Nuclei were counterstained with Hoechst 33342 (Invitrogen). For cell death analysis the In Situ Cell Death Detection Kit for TUNEL (TdT-mediated dUTP nick end labelling) from Roche was used. Bright-field and fluorescence pictures were taken by using the DMI4000 B microscope (Leica) in combination with a LEICA DFC360 FX or LEICA DFC420C camera.
4
Microscopy Imaging of Stained Specimens
5
Microscopic Observation of PHB Granules
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To observe PHB granules within the cells, 100 µL of cyanobacterial cells were centrifuged (1 min at 10,000× g) and 80 µL of the supernatant discarded. Nile Red (10 µL) was added and used to resuspend the pellet in the remaining 20 µL of the supernatant. From these mixtures, 10 µL were taken and applied on an agarose coated microscope slide to immobilize the cells. The Leica DM5500B microscope (Leica, Wetzlar, Germany) was used with a 100× /1.3 oil objective for fluorescence microscopy. To detect Nile red stained PHB granules, an excitation filter BP 535/50 was used, together with a suppression filter BP 610/75. A Leica DFC360FX (Leica, Wetzlar, Germany)) was used for image acquisition.
6
Multimodal Histopathological and Immunofluorescence Analyses
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Histopathological analyses were performed on formalin-fixed paraffin-embedded tissue after Mayer’s haematoxylin and eosin (H&E) staining. Immunofluorescence staining of tissue sections was performed using the Alexa Fluor and TSA Cy3/Fluorescein system as recommended by the manufacturer (Perkin&Elmer). The following antibodies were used: murine MLKL (Biorbyt, Cat.No.: orb32399), GSDMD (abcam, Cat.No.: ab209845), Cytokeratin 19 (abcam, Cat.No.: ab52625), cleaved CASP3 (Cell Signaling, Cat.No.: 9661S), Albumin (abcam, Cat.No.: ab106582), CD11c (BD Bioscience, Cat.No.: 550283), F4/80 (eBioscience, Cat.No.: 14-4801-85), Myeloperoxidase (MPO) (abcam, Cat.No.: ab9535), and secondary anti-rabbit (Dianova, Cat.No.: 111-065-144), anti-chicken (abcam, Cat.No.: ab150169), anti-Armenian hamster (BioLegend, Cat.No.: 405501), anti-rat (Bioscience, Cat.No.: 554014), or anti-rabbit-Alexa 647 (BioLegend, Cat.No.: 406414). Nuclei were counterstained with Hoechst 33342 (Invitrogen). Cell death (TUNEL) was analysed using the in-situ cell death detection kit (Roche). Images were obtained using a confocal fluorescence microscope (LEICA TCS SP5 II) or the microscope LEICA DMI 4000B together with the LEICA DFC360 FX or LEICA DFC420C camera and the imaging software “LAS AF” (Leica). Oil red staining was performed on cryosections as recommended by protocol described before22 .
7
Microscopy Imaging Protocol
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In situ hybridization and immunohistochemical images were taken with a Leica DMNB microscope coupled to a Leica DFC480 camera. Fluorescence images were taken using a Leica DM5500B automated epifluorescence microscope connected to a DFC360FX camera. Image panels were created using Adobe Photoshop CS6.
8
Phenotypic Characterization of Strains in Liquid Culture
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To assess the phenotype in liquid culture of various strains used in this study, cells were grown in 30 ml DNB cultures at 30°C, 250 r.p.m. in flasks with springs to assist aeration. After 16 h, 2 µl of culture was removed and added to the surface of a 1% agarose pad, a coverslip added, and imaged using the 100x DIC Objective of the Leica DM 6000 microscope with Leica DFC360 FX (cooled monochrome) camera. Images were manipulated using the Fiji software package (http://fiji.sc/Fiji ). Prior to assaying BldO‐3xFLAG levels, cultures were grown and prepared in the same way but images captured using a Zeiss Axio Observer.Z1 widefield microscope.
For fluorescence microscopy of the bldOp‐ypet strain (and the strain carrying the empty vector), imaging was conducted using a Zeiss Axio Observer.Z1 widefield microscope. FM® 4‐64 Dye (N‐(3‐Triethylammoniumpropyl)−4‐(6‐(4‐(Diethylamino) Phenyl) Hexatrienyl) Pyridinium Dibromide) (Molecular probes; final concentration 5 µg/ml) was added to samples prior to imaging. DIC images were captured with a 150 ms exposure time, YFP images were captured with a 1000 ms exposure time and RFP images captured with a 250 ms exposure time.
For fluorescence microscopy of the bldOp‐ypet strain (and the strain carrying the empty vector), imaging was conducted using a Zeiss Axio Observer.Z1 widefield microscope. FM® 4‐64 Dye (N‐(3‐Triethylammoniumpropyl)−4‐(6‐(4‐(Diethylamino) Phenyl) Hexatrienyl) Pyridinium Dibromide) (Molecular probes; final concentration 5 µg/ml) was added to samples prior to imaging. DIC images were captured with a 150 ms exposure time, YFP images were captured with a 1000 ms exposure time and RFP images captured with a 250 ms exposure time.
9
Zebrafish Transient Transgenesis Screening
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The following zebrafish lines were used for embryo micro-injection experiments: Slusarski AB (wild-type); egr2b:KalTA4BI-1xUASkCherry (r3r5mCherry)37 (link). Transient transgenic zebrafish embryos were generated for each reporter construct by Tol2-mediated transgenesis in fertilized eggs as described previously38 (link). In general a minimum of 100 embryos were injected to monitor efficiency for each constructs due to mosaicism and position effects of integration. GFP-expressing transient transgenic embryos were raised to adulthood and crossed with either wild-type or r3r5mCherry fish to screen for germline transgene integration38 (link). Embryos were screened for fluorescent reporter expression using a Leica M205FA microscope. Fluorescence and bright-field signals were captured with a Leica DFC360FX camera using LAS AF imaging software. Images were cropped and alterations to brightness and contrast were made using Adobe Photoshop CS5.1.
10
Tick Scutal Index Measurements
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All ticks were subjected to scutal index measurements, except 33 ticks that were not intact anymore. Pictures of every tick were taken with a Leica DFC360FX camera [26 (link)]. On those pictures body length, starting at the basis capituli, and scutum width at the scutums widest point were measured. The scutal index as the quotient of body length and scutum width was calculated [27 (link)].
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