At the previously identified endpoint, cNTs were collected as described [37 (link)] and tissues were fixed in periodate-lysine-paraformaldehyde (PLP) and prepared for sectioning as previously [73 (link)]. Following fixation, cNT were passed through sucrose gradients and frozen in OCT (TissueTek) media. Serial sections (7 μm) were cut using a cryostat. Prior to staining, all slide-mounted tissue sections were blocked with PBS containing 1% BSA, 0.1% Tween-20, and 10% rat serum. Sections were stained with the following antibodies: anti-Streptococcus pyogenes Group A Carbohydrate (Abcam, ab9191), anti-B220 (Biolegend, RA3-6B2), anti-CD3 (Biolegend, 17A2), and anti-Ly6G (Biolegend, 1A8). After staining, sections were mounted with ProLong Gold Antifade Reagent (Invitrogen). Tiled images of whole cNT sections were collected using a DM5500B fluorescence microscope (Leica) at 10× and 20×.
Dm5500b fluorescence microscope
Manufactured by Leica
Sourced in Germany, United States
The Leica DM5500B is a fluorescence microscope designed for advanced imaging applications. It features a high-resolution optical system and a range of illumination options, including LED and mercury vapor lamps, to enable a variety of fluorescence techniques. The DM5500B is capable of capturing high-quality images and offers versatile configuration options to meet the specific needs of researchers and scientists.
Automatically generated - may contain errors
Lab products found in correlation
Prolong gold antifade reagent, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Anti b220, by BioLegend (1 mentions)
Anti cd3, by BioLegend (1 mentions)
Anti ly6g, by BioLegend (1 mentions)
Ms excel 2013, by GraphPad (1 mentions)
Prism 5, by GraphPad (1 mentions)
Il md led inverted fluorescence microscope, by Leica (1 mentions)
Anti his alexa488, by Qiagen (1 mentions)
Mikrom hm 650 5, by Thermo Fisher Scientific (1 mentions)
Dfc360 fx camera, by Leica (1 mentions)
Goat anti rabbit alexa 555, by Thermo Fisher Scientific (1 mentions)
Fluoro carbonyl cyanide phenylhydrazone (fccp), by Cayman Chemical (1 mentions)
Jc 1 dye, by Adipogen (1 mentions)
Floid cell imaging station, by Thermo Fisher Scientific (1 mentions)
Geltrex, by Thermo Fisher Scientific (1 mentions)
Mitotracker cmx red, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Jackson ImmunoResearch (1 mentions)
Noble agar, by Merck Group (1 mentions)
31 protocols using dm5500b fluorescence microscope
1
Immunohistochemical Analysis of cNT Sections
2
Macrogamete and Zygote Formation Analysis
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For the analysis of macrogamete and zygote formation, mature gametocytes of WT NF54, the 7-Helix-1-KO lines 2E6 and 1D12 and the complementation line 7-Helix-1-KO(+) were enriched via Percoll purification and set to a gametocytemia of 2–4%. For inhibitor experiments, the parasites were incubated with emetine or cycloheximide at IC50 concentration (as determined by Malstat assay) for 30 min at 37°C. Incubation with the solvents alone was used as a negative control. The gametocytes were activated and at 30 min p.a. (macrogametes) or 4 h p.a. (zygotes), the samples were subjected to IFA. Immunolabeling was performed as described above and macrogametes and zygotes were labelled with rabbit anti-Pfs25 antisera. Parasites were counted microscopically using a Leica DM 5500B fluorescence microscope with 600-fold magnification. For comparative macrogamete and zygote formation assays, 30 optical fields were counted for three to four times. The relative numbers of macrogametes and zygotes were calculated (WT set to 100%). Three to eight independent experiments were conducted; data analysis was performed using MS Excel 2013 and GraphPad Prism 5. For inhibitor experiments, macrogametes and zygotes per 1,000 RBC were counted for five times. Two independent experiments were performed; data analysis was performed using MS Excel 2013 and GraphPad Prism 5.
3
HSV-1 Fluorescent Viral Dynamics
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We used a genetically engineered HSV-1 construct incorporating enhanced green fluorescent protein (EGFP) and monomeric red fluorescent protein (RFP) as reporter genes whose expression is driven by the viral promoters ICP0 and Glycoprotein C, respectively.25 (link) EGFP expression in infected cells indicates that HSV-1 has entered lytic cycles, while RFP expression indicates commitment to viral DNA replication. Cells were incubated with HSV-1 for 2 h at specified multiplicity of infection (MOI). To inhibit viral replication, cells were preincubated with antivirals used in animal models: acyclovir (50 μM), or (E)-5-(2-bromovinyl)-2′-deoxyuridine (5BVdU, 30 μM) along with interferon-alpha (IFN-α, 125 U/ml).22 (link) The proportion of cells expressing EGFP and RFP was determined by flow cytometry (FC). Images were acquired using a Leica IL MD LED inverted fluorescence microscope and a Leica DM5500B fluorescence microscope.
4
Quantifying Cell-Specific CEA Binding
5
Spinal Cord and Lymph Node Preparation
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Spinal cords and lymph nodes were extracted from mice and prepared, as previously described (25 (link)). Briefly, whole lymph nodes and spinal cords were fixed in periodate–lysine–paraformaldehyde (PLP) and subsequently passed through sucrose gradients to protect from freezing artifacts. Lymph nodes were frozen whole in OCT (TissueTek) media. Spinal cords were cut into five to nine evenly spaced pieces and arranged in order prior to freezing in OCT. Serial cryostat sections (7 μm) were blocked in PBS containing 1% Bovine Serum Albumin, 0.1% Tween-20, and 10% rat serum before proceeding with staining. Sections were mounted with ProLong Gold Antifade Reagent (Invitrogen) and stored at −20°C. Tiled images of whole spinal cord sections (20×) were imaged using DM5500B fluorescence microscope (Leica).
6
Histological Sectioning and Neural Tracing
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For histology, brain samples were embedded in 4% agarose and sectioned into 100 μm slices using a vibratome (Mikrom HM 650 V, Thermo Scientific). Nerve and whisker samples were prepared for cryo-sectioning by embedding in 10% sucrose and 4% agarose and then left in 30% Sucrose solution until they sunk to the bottom for cryo-protection. Samples were then sectioned into 40–60 μm slices at −15°C using a Leica Frigomobil 0247/08.1996 cryo-sectioning device.
Sections were immediately mounted on gelatine-coated slides with 0.1 M PB and imaged using a Leica DM5500B fluorescence microscope (Wetzlar, Germany). For neural tracing with Carbocyanine dyes, immediate imaging after sectioning is preferred as the dye continues to diffuse through the tissue. Leaving sections for too long can result in leaking of the dye out of the cut cells leading to diffuse and non-specific labeling. After imaging, the sections can be kept in 0.1 M PB for further histological staining.
Sections were immediately mounted on gelatine-coated slides with 0.1 M PB and imaged using a Leica DM5500B fluorescence microscope (Wetzlar, Germany). For neural tracing with Carbocyanine dyes, immediate imaging after sectioning is preferred as the dye continues to diffuse through the tissue. Leaving sections for too long can result in leaking of the dye out of the cut cells leading to diffuse and non-specific labeling. After imaging, the sections can be kept in 0.1 M PB for further histological staining.
7
Immunofluorescence Staining of eNOS
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Paraffin sections were deparaffinized. Antigen retrieval was performed for 15 min in citrate buffer using a rice cooker, then slides were cooled down on ice for 20 min. After washing slides three times in TBST (TBS buffer + 0.1% Tween 20) for 5 min, slides were blocked with 3% bovine serum albumin (BSA) and 0.4% Triton X-100 [in Tris-buffered saline (TBS)] at room temperature (RT) for 1 h and then incubated with primary antibody against eNOS (ThermoFisher Scientific, PA1-037; 1:100) at 4 °C overnight. Afterwards, slides were again washed three times in TBST for 5 min, incubated with secondary antibody (Goat Anti-Rabbit Alexa 555, ThermoFisher Scientific, A-21429; 1:500) in room temperature for 1 h, and then washed three times in TBST before being mounted with Prolong Gold Anti-fade Reagent with DAPI (4,6-diamidino-2-phenylindole; ThermoFisher Scientific, P36931). Fluorescent images were acquired using Leica DM5500 B fluorescence microscope connected to Leica DFC360 FX camera.
8
Mitochondrial Dynamics in Cancer Stem Cells
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To evaluate mitochondrial membrane depolarization, transduced CRC-SC#1 cells were plated at a concentration of 30,000 cells/well in a 48-well plate pre-coated with Geltrex (Thermo Fisher Scientific, Waltham, MA, USA). Cells were stained with 10 μg/ml JC-1 dye (Adipogen, San Diego, CA, USA) in PBS for 30 min in the dark at 37 °C. FCCP (Cayman Chemicals, Ann Arbor, MI, USA) was added for 15 min at the end of the staining as a positive control. Signals were acquired with a fluorescence microscope (FLoid Cell Imaging Station, Life Technology) and images were analyzed by ImageJ software to determine the red/green fluorescence ratio. Mitochondrial morphology was assessed through Mitotracker CMX-Red (Invitrogen, Waltham, MA, USA). CRC-SC#1 cells were seeded at a concentration of 100,000 cells/well in a 24-well plate pre-coated with Geltrex. Subsequently, cells were stained with 10 nmol/L Mitotracker for 30 min at 37 °C. Fluorescence was visualized by DM5500B fluorescence microscope (Leica, Wetzlar, Germany), and analyzed using ImageJ software v 1.52a (Ashland, OR, USA).
9
Fluorescent Imaging of Blastocyst
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FAM-labeled tDR-14:32-Glu-CTC-1 agomir scramble control was purchased from GenePharma (Shanghai, China) and supplemented into the conditioned medium of group cultured presumed zygotes on 1 dpi. On 8 dpi, blastocysts were washed with 1 mg PVP/mL PBS for three times and then fixed in 4% paraformaldehyde under ambient temperature for 1 h. After fixing, blastocysts were stained with Hoechst 33342 (Thermo Fisher, Waltham, MA, USA) for 10 min and imaged by Leica DM5500B Fluorescence Microscope. Images were merged by ImageJ 8.0.
10
Drosophila Retinal Development Protocol
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Wild-type and mutant stocks were maintained on nutrient-rich Drosophila media at 25°C. Pre-pupae were selected and maintained in humidified chambers until dissection at 40 h after puparium formation (APF) (DeAngelis and Johnson, 2019 (link)). Rabbit anti-Cno (1:500) and chicken anti-GFP (1:8000, Abcam #13970) followed by Alexa-Fluor-488-conjugated secondary antibodies (Jackson ImmunoResearch #711-545-152 or #703-545-155) were used to detect Cno, CnoWT-GFP and cnoΔDIL-GFP, and retinas imaged with a Leica DM5500 B fluorescence microscope. We performed dissections in triplicate, with 5–10 pupae of each genotype dissected each time. Patterning errors were scored in retinas from one representative dissection of three carried out in triplicate, as previously described (Johnson and Cagan, 2009 (link)). Analyses spanned 9–15 eyes for each genotype, with 110 data points (ommatidia) per genotype. Significance was assessed using unpaired t-tests with Welch’s correction (two-tailed P-value). Image files were processed for publication using Adobe Photoshop.
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