Tissue samples were fixed overnight in 4% PFA, rinsed in 1x phosphate-buffered saline (PBS). Post-embryonic samples were decalcified in Osteosoft (Merck, 1017281000). Samples were dehydrated before paraffin embedding and sectioned at 7 μm. For hematoxylin/eosin/alcian blue staining, sections were initially treated in a solution of 1% Alcian blue in 3% acetic acid before classical hematoxylin/eosin staining. For ‘critical electrolyte concentration’ applications (Scott and Dorling, 1965 (link)), the initial treatment was replaced by 0.05% alcian blue and 0.6M MgCl2 in 0.2M acetate buffer. Sirius Red staining were performed in 0.1% Direct Red 80 (Sigma, 365548) in 1.2% picric acid and rinsed in 0.5% acetic acid. Elastic staining was performed following the manufacturer’s instructions (elastic stain kit, Sigma HT25A-1KT). Images were acquired with a Pannoramic MIDI Slide scanner (3D HISTECH, Budapest, Hungary). Polarised images were taken with a Leica DM5500 microscope.
Dm5500 microscope
Manufactured by Leica camera
Sourced in Germany
The Leica DM5500 is a high-performance microscope designed for advanced laboratory applications. It features a robust and ergonomic design, providing a stable platform for precise observations and measurements. The DM5500 offers a range of optical configurations, including brightfield, darkfield, and phase contrast, enabling diverse imaging capabilities to support various research and analytical needs.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (4 mentions)
Rolera mgi camera, by Leica camera (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Pe 100 ex 770 nm cool led, by Leica Microsystems (2 mentions)
Indocyanine 775 845 nm filter, by Leica Microsystems (2 mentions)
In situ cell death detection kit, by Roche (2 mentions)
Metamorph software, by Molecular Devices (2 mentions)
Ab5076, by Abcam (2 mentions)
Las software, by Leica camera (2 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (2 mentions)
Direct red 80, by Merck Group (1 mentions)
Elastic stain kit, by Merck Group (1 mentions)
Pannoramic midi slide scanner, by 3DHISTECH (1 mentions)
Osteosoft, by Merck Group (1 mentions)
Superfrost plus, by Thermo Fisher Scientific (1 mentions)
Reichert ultracut s, by Leica camera (1 mentions)
Toluidine blue o, by Merck Group (1 mentions)
Axio imager m2, by Zeiss (1 mentions)
Anti 53bp1, by Cell Signaling Technology (1 mentions)
Dako fluorescent mounting medium, by Agilent Technologies (1 mentions)
55 protocols using dm5500 microscope
1
Histological Analysis of Tissue Samples
2
Quantification of Photoreceptor Nuclei and Retinal Layer Thickness
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Mouse eyes were fixed in 4% PFA, cryoprotected with 30% sucrose, embedded in OCT and cryosectioned. Fourteen‐micrometer cryosections was collected on slides (Superfrost Plus; Fisher Scientific, Pittsburgh, PA), and washed three times with PTW1×. The sections were then incubated with DAPI in phosphate‐buffered saline (PBS)1× for 10 min. Count of nuclei in ONL was performed on three eyes/genotype (at least two sections per eye) at p30. Only sections in which the optic nerve was contained were used, and two images from each section were photographed under a Leica DM‐5500 microscope, with the objective Leica ∞/0.17/D, HCX PL FLUOTAR, 40×/0.75 that acquires an area of 0.31 mm2, in comparable regions equidistant from the optic nerve head (Barbato et al, 2017 (link); Karali et al, 2020 (link); Ciampi et al, 2022 (link); Intartaglia et al, 2022 (link)). The acquired images were used for nuclei counts. DAPI‐positive nuclei in ONL were count manually using ImageJ program and normalized per area. On the same images, the ONL thickness was manually measured using ImageJ program, by measuring the distance between the Henle's fiber layer (HFL) and the outer limiting membrane (OLM). The same analysis was performed at p90 on N ≥ 3 eyes/genotype (at least two sections per eye).
3
Quantification of Retinal Apoptosis
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Immunostained sections for Active‐casp3, in which the optic nerve was contained, were analyzed with a Leica DM‐5500 microscope and positive cells were manually counted and normalized per section. The analysis was performed on 4–6 eyes/genotype on at least eight sections/eye that contain the optic nerve.
4
Labeling Caenorhabditis elegans neurons with DiI
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A stock dye solution prepared in DMSO containing 5 mg/ml red fluorescent lipophilic dye DiI (Sigma, Cat. 42364) was diluted in M9 buffer to a final concentration of 5 μg/ml for optimal signal intensity. Animals carrying the srh-234p::GFP reporter (VDL3) were soaked in DiI for 1 h and then rinsed with M9 buffer twice. Stained animals were recovered for 1 h on NGM plates seeded with either E. coli OP50 or Comamonas aq. DA1877 before examination of dye-filled ADL neurons with a Leica DM5500 microscope equipped with epifluorescence.
5
Quantifying Ventral Nerve Cord Growth Cones
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VD growth cones were imaged and quantified as previously described [22 (link)]. Briefly, animals at ~16 h post-hatching at 20°C were placed on a 2% agarose pad and paralyzed with 5mM sodium azide in M9 buffer, which was allowed to evaporate for 4 min before placing a coverslip over the sample. Some genotypes were slower to develop than others, so the 16 h time point was adjusted for each genotype. Growth cones were imaged with a Qimaging Rolera mGi camera on a Leica DM5500 microscope. Images were analyzed in ImageJ, and statistical analyses done with Graphpad Prism software. As described in [22 (link), 23 (link)], growth cone area was determined by tracing the perimeter of the growth cone body, not including filopodia. Average filopodial length was determined using a line tool to trace the length of the filopodium. Unless otherwise indicated, ≥25 growth cones were analyzed for each genotype. These data were gathered in ImageJ and entered into Graphpad Prism for analysis. Analysis of Variance (ANOVA) was used to determine significance of difference between genotypes. Any of the VD growth cones visible at the time of imaging were scored (VD2-VD13), and we did not focus on any single VD growth cone for analysis.
6
Tissue Sectioning and Staining Protocol
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For cross sectioning, tissues were dissected and fixed with 2% glutaraldehyde in 50 mM PIPES buffer, pH 6.9 overnight. Samples were dehydrated in graded ethanol series and embedded in Technovit 7100 resin (Heraeus) according to the manufacturer's protocol. Sections of 2 uM were cut using an ultramicrotome (Reichert Ultracut S, Leica) and stained with 0.05% wt/vol toluidine blue O (Sigma-Aldrich) for 20 s. All samples were rinsed in tap water for 30 s to 1 min. After drying, the sections were mounted in DePex medium (EMS, Hatfield, PA) and covered with cover slips. Sections on slides were analyzed with a Leica DM5500 microscope.
7
Quantifying Neuronal Activity Markers
8
Quantitative Retinal Cell Imaging
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Histologic and in situ hybridization sections were imaged with a Zeiss Axio Imager M.2 microscope, color camera and Zen software (v2.6). Antibody-labeled cryosections were imaged using a Leica DM5500 microscope, equipped with a SPEII solid state laser scanning confocal and processed using Leica LASX (v.5) plus Navigator tiling subprogram, FIJI/Image J Software (NIH) and Adobe Photoshop (CS5) software programs. All images were equivalently adjusted for brightness, contrast, and pseudocoloring. At least 3 biologic replicates per age and genotype were analyzed for each marker, and at least 2 sections per individual were quantified via cell counting or tissue area measurements. Sections were judged to be of equivalent depth by presence of or proximity to the optic nerve. For E17 retinal sections, 11 tile scanned retinal sections from 2 biologic replicates/genotype were quantified. Marker+ cells in tissue sections were counted using the count tool in Adobe Photoshop CS5 and statistical analyses performed using Prism (GraphPad v9) or Excel (v16.16.11) software, with p-values determined with oneway ANOVA and pair-wise Dunnett’s test. A p-value less than 0.05 was considered statistically significant.
9
Hypoxia-Induced DNA Damage Quantification
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A549 cells were preincubated in hypoxic conditions (0.2% O2) for 20 hours, followed by treatment with evofosfamide for 4 hours under hypoxic conditions, reoxygenation and irradiation with 2 Gy. 20 hours after irradiation, cells were washed twice with PBS, fixed with 4% formaldehyde/PBS for 10 min, and washed with PBS (4 × 5 min). Cells were then permeabilized for 5 min with 0.2% ice cold Triton-X-100/PBS, blocked for at least 20 min with 1% BSA, followed by 1 hour incubation with the rabbit monoclonal anti-H2AX-pSer139 (1:100, Abcam, Cambridge, UK) or the rabbit polyclonal anti-53BP1 (1:200, Cell Signaling, Boston MA, USA) primary antibodies, diluted in 1% BSA/PBS. After washing with 1% BSA/PBS (3 × 15 min), cells were incubated with the appropriate secondary antibody diluted 1:1000 (Alexa-488), washed with 1% BSA/PBS (2 × 10 min) followed by PBS (1 × 10 min) and incubated with DAPI/Methanol (1 μg/ml) for 3 min, before fixation with Dako Fluorescent Mounting Medium (Dako, North America). Images were taken using a Leica DM 5500 microscope at a magnification of 40x and quantified using FociCounter software. At least 50 cells/condition were analyzed.
10
Immunohistochemical Analysis of Prostate Tumors
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Prostates with tumors were fixed overnight at 4 °C with 4% paraformaldehyde. Then the tissues were dehydrated and embedded in paraffin. Sections (4 μm) were used for hematoxylin/eosin/safran (HES), scFvD2B and scFvD2BGF7.7 staining after dewaxing and rehydration in alcohol. The scFv fragments (scFvD2B and scFvD2BGF7.7) were detected using anti-Histidine antibody HIS-H8 (Pierce). The DAKO Envision FLEX detection system included pretreatment with DAKO PT Link (pre-treatment module) and target retrieval solution, pH 6 (K800521) 97 °C for 20 min. All steps of immunostaining were done in an automated immunostainer (Autostainer Plus, DAKO, Les Ulis, France). Inhibition of endogenous peroxidases (H2O2 3% in water, 10 min) was followed by incubation of the primary antibody (1/250, 45 min). Sections were incubated with HRP conjugated Envision (DAKO, 20 °C, 20 min) and revealed with DAB. Images were obtained on Nikon Eclipse 50i using NIS-element F software. Fluorescence from X770 was also detected on the processing slices using Leica DM 5500 microscope fitted with pE-100 (Ex 770 nm) Cool LED and a indocyanine (775/845 nm) filter (Leica Microsystems).
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