Transgenic worms carrying GFP reporters were imaged using either an Olympus BX51 fluorescence microscope or a Nikon A1R confocal microscope. GFP levels were determined using NIS Elements software, and an ANOVA was used to assess significance. The sizes of ER patches were determined using Image J, and the Fisher exact test was used with Bonferroni correction. Adobe Photoshop was used to assemble plates for figures. The ER cortical enrichment ratios were determined using Image J. The second-most proximal oocyte was measured in five fog-2; GFP::SP12 worms and five GFP::SP12 worms. Six line intensity scans were drawn in the dorsal-ventral and the anterior-posterior orientations, and averaged. For each orientation, the cortical enrichment ratio was calculated by dividing the average for the arrested oocytes by the average for the non-arrested oocytes. A ratio greater than 1.0 indicates cortical enrichment in arrested oocytes.
Bx51 fluorescence microscope
Manufactured by Nikon
The BX51 fluorescence microscope is a high-performance microscope system designed for advanced microscopy applications. It features a stable, vibration-resistant body, a high-intensity illumination system, and a range of optical configurations to support a variety of fluorescence imaging techniques.
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5 protocols using bx51 fluorescence microscope
1
ER Cortical Enrichment in Arrested Oocytes
2
Immunocytochemical Profiling of Cell Surface and Intracellular Antigens
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Cells were cultured on glass coverslips in the wells of 24-well plates for at least 48 h, and then fixed in buffered 4% paraformaldehyde for 10 min and washed in PBS, pH 7.4. To detect surface antigens, after blocking with 5% normal goat serum in PBS, cells were incubated with antibodies targeting O4 sulfatide, O1/galactocerebroside (GalC), NG2, p75NTR or A2B5 without permeabilization for 90 min at room temperature. For the immunostaining of intracellular antigens, cells were post-fixed and permeabilized with ethanol: acetic acid (19:1) for 10 min at −20°C and then incubated with the respective primary antibodies overnight at 4°C. The next day, the cells were incubated with anti-mouse, anti-rat, or anti-rabbit antibodies conjugated to Alexa fluorochromes (1:500 dilution) for 30–45 min at room temperature in the dark. Both primary and secondary antibodies were diluted in blocking solution. Nuclei were counterstained with bisBenzimide hydrochloride (Hoechst 33342, 3 × 10–5 M) in PBS for 5 min. The coverslips were rinsed in distilled water and mounted on slides with Prolong Gold. The samples were analyzed and imaged using an Olympus BX51 fluorescence microscope or a Nikon Eclipse Ti confocal microscope. The list of antibodies and suppliers is shown in Supplementary Table S5 .
3
Bacterial Morphological Characterization
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Isolates were cultured on LB agar at 37°C for 24 h, and their morphological traits were observed. Gram’s staining and endospore staining by Schaeffer–Fulton method was performed as described by Benson (2002) and Bartholomew and Mittwer (1950) (link). The shape and size of the bacteria were observed with a Nikon BX-51 fluorescence microscope and an SU-8010 scanning electron microscope.
4
PDGF and Fibronectin Regulate SMC Proliferation
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Human and mouse SMCs were serum-starved for 48 hours to induce quiescence. Cells were washed and stimulated with PDGF-BB (20 ng/mL), cFn (20 μg/mL) or pFn (20 μg/mL), and recombinant EDA-containing or EDA-lacking peptides (10 μg/mL) for 24 hours. Cells were incubated with 10 μM BrdU for the last 12 hours of treatment (50 (link)). Carotid artery sections from BrdU-treated mice or cultured SMCs were fixed with 4% PFA (in PBS). DNA hydrolysis was performed by treating the cells/sections with 2 M HCl for 20 minutes. Samples were subjected to immunofluorescence staining with a mouse monoclonal anti-BrdU antibody (1:200, Abcam) together with αSMA (1:200, MilliporeSigma) for 3 hours at 37°C and labeled with appropriate Alexa Fluor 488 and Alexa Fluor 546 secondary antibodies (1:400, Abcam). Nuclei were stained with Hoechst (5 μg/mL) before mounting. Images of the sections were acquired using an Olympus BX51 fluorescence microscope, and images of cells were acquired using the Nikon Eclipse Ti-U fluorescence microscope. The number of BrdU-positive cells in the neointima or the media and the total number of nuclei were determined using ImageJ software. The percentage of BrdU-positive cells was calculated as (number of BrdU-positive nuclei in the neointima or media/total number of nuclei) × 100.
5
Fusarium Hyphal Morphology Analysis
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A thin layer of PDA medium was poured onto a sterile plate, and a sterile glass slide was placed in the center of the plate after the medium had solidified. Then approximately 200 μL of PDA medium was spread onto the glass slide, and a sterile 1-mL pipette tip was used to place a Fusarium cake onto the slide. An Oxford cup was placed at each end of the slide; 200 μL of cell-free culture filtrate was added to one cup as the treatment, and sterile distilled water was added to the other cup as the control. After incubating for 3 days, the slide was observed under a Nikon BX-51 fluorescence microscope. The hyphae at the edge of the bacteriostatic area were picked with an inoculation needle and placed in a sterile centrifuge tube that contained 2.5% glutaraldehyde fixative for 24 h. The control area was sampled similarly, and the samples were sent to Keshang Biotech Co., Ltd., for scanning electron microscope observation.
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