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).
Leica dfc360 fx
Manufactured by Leica camera
Sourced in Germany
The Leica DFC360 FX is a digital camera designed for microscopy applications. It features a high-resolution sensor and the ability to capture images and videos at various exposure times and frame rates.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Spss 23, by IBM (1 mentions)
Leica dm6000b, by Leica Microsystems (1 mentions)
Filipin, by Merck Group (1 mentions)
Dm5500b, by Leica camera (1 mentions)
Dmi4000b microscope, by Leica camera (1 mentions)
Anti cleaved caspase 8, by Cell Signaling Technology (1 mentions)
Biotinylated anti rabbit secondary antibody, by Dianova (1 mentions)
Anti β catenin, by Cell Signaling Technology (1 mentions)
Tsa cy3 system, by PerkinElmer (1 mentions)
Anti villin, by Santa Cruz Biotechnology (1 mentions)
Anti caspase 8, by Cell Signaling Technology (1 mentions)
Leica dm6000b, by Leica camera (1 mentions)
Leica dm5500b microscope, by Leica camera (1 mentions)
Albumin, by Abcam (1 mentions)
Cytokeratin 19, by Abcam (1 mentions)
Las af, by Leica camera (1 mentions)
Anti chicken, by Abcam (1 mentions)
Cleaved casp3, by Cell Signaling Technology (1 mentions)
Anti armenian hamster, by BioLegend (1 mentions)
6 protocols using leica 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 .
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