Slides were incubated in PBT (0.15% BSA, 0.1% Tween 20 in PBS) for 60 min prior to overnight incubation at room temperature with the antibodies listed in supplementary material Table S1 . Thereafter, slides were washed 3 times for 5 min each in PBS with 0.1% Tween 20, incubated with secondary antibodies (Invitrogen or Jackson ImmunoResearch) at 1:500 for 60 min in PBT, washed in PBS plus 0.1% Tween 20, and mounted in Vectashield with DAPI. Images of germ cells were acquired with an ECLIPSE Ti-E microscope (Nikon) and Zyla 5.5 sCMOS camera (Andor Technology), with 20×, 60×, and 100× CFI Apochromat TIRF oil immersion lenses (Nikon), numerical aperture 1.40; image acquisition was performed using NIS-Elements Basic Research software (Nikon). Images were taken at RT (∼22°C). Phylum, Volocity 3D Image Analysis (PerkinElmer), NIS-Elements Basic Research, and NIS-Elements Viewer (Nikon) were used for image analysis. Photoshop and Illustrator (Adobe) were used for composing figures. Particular stages of primary spermatocytes were determined by staining for H2AX, SYCP3 and/or H1T. For data analysis, the matched substage of meiosis was analyzed in controls and mutants. All data were confirmed with at least two or three independent mice. Total numbers of analyzed nuclei in at least two independent experiments are shown in each panel.
Nis elements basic research
Manufactured by Nikon
Sourced in Japan, United States
NIS-Elements Basic Research is a software for microscopy imaging and analysis. It provides a platform for controlling microscope hardware and capturing digital images. The software offers tools for image processing, measurement, and data management.
Automatically generated - may contain errors
Lab products found in correlation
Nis elements viewer, by Nikon (2 mentions)
Zyla 5.5 scmos camera, by Oxford Instruments (2 mentions)
Cfi apochromat tirf oil immersion lenses, by Nikon (2 mentions)
Phylum, by PerkinElmer (2 mentions)
Volocity 3d image analysis, by PerkinElmer (2 mentions)
Eclipse ti e microscope, by Nikon (2 mentions)
Ds ri2 digital camera, by Nikon (1 mentions)
Eclipse ti u, by Nikon (1 mentions)
Mathematica, by Wolfram (1 mentions)
Anti kv11, by Alomone (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Eclipse 80i, by Nikon (1 mentions)
Te2000 e microscope, by Nikon (1 mentions)
Nis elements basic research software, by Nikon (1 mentions)
Eclipse 80i light microscope, by Nikon (1 mentions)
Sa00009 2, by Proteintech (1 mentions)
Hoechst 33342, by Beyotime (1 mentions)
Eclipse 50i, by Nikon (1 mentions)
13 protocols using nis elements basic research
1
Immunofluorescence Imaging of Meiotic Germ Cells
2
Quantifying Vascular Density and Smooth Muscle
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The vascular bed was fixed with 4% formalin (Muto Pure Chemicals), and three sections were prepared randomly in the direction perpendicular to the SFA and SFV. Immunofluorescence staining was performed using primary antibodies against αSMA and CD31 (see below). Four random images were taken at high magnification (×40 objective), and the luminal structures that stained positively for both αSMA and CD31 were counted as blood vessels. The number of blood vessels was measured, and blood vessel density was determined. Vascular smooth muscle area was calculated using NIS-Elements Basic Research (Nikon). All measurements were made in a double-blind manner.
3
Fern-Like Mucin Crystallization Analysis
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It has been shown that drying of mucosal fluids in presence of Na and K ions forms fern-like patterns due to crystallization of mucins (glycosylated proteins) which are easily observable by optical microscopy (Fern-Test). We applied a similar method to the nasal mucus to validate our sampling method by confirming the presence of mucin and by evaluating its quality. A pool at 2 g/l of the mucus of 10 newborn rabbits was sampled and diluted at 1/10 in DPBS x1 or H2O (negative control) and were deposited on a glass slide (3 × 5 µl drops) and controls with bovine mucin at 2 g/l, diluted at 1/10 in DPBS x 1 or H2O, were also realized. Another control with only DPBS x 1 (no mucus) was carried out in the same conditions. After about 4 h of drying at 37 °C, the slides were examined with a microscope Eclipse E600 equipped with a 4x plan fluor objective. Images (see Supplementary Fig. S4) were acquired with a Ds-Ri2 digital camera using the software Nis-Elements Basic Research (all from Nikon, Tokyo, Japan). The absence of blood in the samples was checked using NanoDrop spectrophotometer ND 1000 technology.
4
Evaluating DNA/PEI Transfection Efficiency
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To determine whether the released DNA/PEI nanocomplexes may be applied for transfection, an in vitro experiment was performed. After preparing PEMs in wells, DMEM with 10% of FBS was filled without bubbles for electrical field treatment. After a release for 48 h, the supernatants mixed with 12,500 of NIH/3T3 cells were seeded per well. The expression of pEGFP-C3 from transfected cells was examined using fluorescence microscopy (Eclipse Ti-U, Nikon, Tokyo, Japan). The intensities of fluorescence were quantified using an image software (NIS Elements Basic Research, Nikon, Tokyo, Japan).
5
TIRFM Imaging of Supported Lipid Bilayers
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TIRFM experiments were conducted
at 22 ± 0.5 °C on a Nikon A1R TIRF instrument and a home-built,
objective-based TIRFM instrument as described previously.57 (link)−59 (link) Supported lipid bilayers in physiological buffer [140 mM KCl, 15
mM NaCl, 0.5 mM MgCl, 26 μM CaCl2, 20 μM EGTA,
5 mM reducedl -glutathione, and 25 mM HEPES (pH 7.4)] were
imaged before and after sequential additions of buffer, BSA, and fluorescent
protein. Typically, only a few dim, rapidly dissociating fluorescent
contaminants were observed on the bilayer prior to the addition of
protein. After protein addition, samples were allowed to equilibrate
for 5 min. Then, to minimize contributions from small numbers of immobile
fluorescent particles (presumably inactive protein aggregates), a
bleach pulse power ∼30-fold higher than that used for imaging
was applied for ∼5 s, and the fluorescence was allowed to recover
for 60 s before data were acquired. For each sample, a set of three
or four movie streams were acquired at a frame rate of 20 frames/s,
and a spatial resolution of 6.3 pixels/μm on the Nikon A1R instrument
or 4.2 pixels/μm on the home-built instrument, using NIS Elements
Basic Research (Nikon). Subsequent particle tracking analysis was
conducted using ImageJ,64 (link) and data processing
and fitting were conducted using Mathematica (Wolfram Research) and
GraphPad Prism 5 (GraphPad Software, Inc.).
at 22 ± 0.5 °C on a Nikon A1R TIRF instrument and a home-built,
objective-based TIRFM instrument as described previously.57 (link)−59 (link) Supported lipid bilayers in physiological buffer [140 mM KCl, 15
mM NaCl, 0.5 mM MgCl, 26 μM CaCl2, 20 μM EGTA,
5 mM reduced
imaged before and after sequential additions of buffer, BSA, and fluorescent
protein. Typically, only a few dim, rapidly dissociating fluorescent
contaminants were observed on the bilayer prior to the addition of
protein. After protein addition, samples were allowed to equilibrate
for 5 min. Then, to minimize contributions from small numbers of immobile
fluorescent particles (presumably inactive protein aggregates), a
bleach pulse power ∼30-fold higher than that used for imaging
was applied for ∼5 s, and the fluorescence was allowed to recover
for 60 s before data were acquired. For each sample, a set of three
or four movie streams were acquired at a frame rate of 20 frames/s,
and a spatial resolution of 6.3 pixels/μm on the Nikon A1R instrument
or 4.2 pixels/μm on the home-built instrument, using NIS Elements
Basic Research (Nikon). Subsequent particle tracking analysis was
conducted using ImageJ,64 (link) and data processing
and fitting were conducted using Mathematica (Wolfram Research) and
GraphPad Prism 5 (GraphPad Software, Inc.).
6
Immunohistochemical Analysis of hERG Channels
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HEK-hERG cells were cultured on ∅ 15 mm glass coverslips coated with 0.1% gelatin. After 24 h treatment with pentamidine (10 μM), dofetilide (1 μM) or 1, 2 or 5 (10 μM) coverslips were washed with PBS++, fixated with 3%-paraformaldehyde and permeabilized with 0.5% Triton X-100 (in PBS). After permeabilization cells were quenched with PBS/glycine (50 mM) and incubated twice with NET-gel (0.25% gelatin, 50 mM Tris–Cl, 150 mM NaCl, 4 mM EDTA, 0.05% Igepal, ±0.01% NaN3, pH 7.4). Cells were incubated with the primary antibody in NET-gel overnight (anti-Kv11.1, Alomone Labs, Jerusalem, Israel). After washing the cells, samples were incubated with the secondary antibody (Jackson). Coverslips were mounted with Vectashield (Vector Laboratories Inc., Burlingame, USA) and imaged using a Nikon Eclipse 80i (Nikon, Amsterdam, The Netherlands) and NIS elements Basic Research (Nikon, Amsterdam, The Netherlands) software.
7
Microscopy Techniques for Germ Cell Analysis
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Images of germ cells were acquired with one of two microscope systems: (1) A TE2000-E microscope (Nikon) and CoolSNAPHQ camera (Photometrics), with 60× and 100× Apochromat oil immersion lenses (Nikon), numerical aperture 1.40; image acquisition was performed using Phylum software (PerkinElmer); (2) an ECLIPSE Ti-E microscope (Nikon) and Zyla 5.5 sCMOS camera (Andor Technology), with 60× and 100× CFI Apochromat TIRF oil immersion lenses (Nikon), numerical aperture 1.40; image acquisition was performed using NIS-Elements Basic Research software (Nikon). Images were taken at RT (∼22°C). Phylum, Volocity 3D Image Analysis (PerkinElmer), NIS-Elements Basic Research, NIS-Elements Viewer (Nikon), and ImageJ (National Institutes of Health) were used for image analysis. Photoshop and Illustrator (Adobe) were used for composing figures. Particular stages of primary spermatocytes were determined by staining for SYCP3 and H1t. XY axes were distinguished by SYCP3 staining if γH2AX was severely disrupted. For data analysis, the matched substage of meiosis was analyzed in controls and mutants. All data were confirmed with at least two or three independent mice. Total numbers of analyzed nuclei in at least two independent experiments are shown in each panel.
8
Microscopic Evaluation of Emulsion Microstructure
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A Nikon Eclipse 80i light microscope (New York, NY, USA) coupled with a Nikon charge-coupled device (CCD) camera (Kanagawa, Japan) and digital image processing software (NIS-Elements Basic Research, Nikon Instruments, New York, NY, USA) were used to observe the microstructure changes of the emulsion samples. Prior to observation under the microscope, the emulsion sample was gently dropped on a microscope slide and covered with a cover slip. The micrographs of the emulsion samples were observed and captured at a magnification of 60×.
9
Quantifying Interlobular Connective Tissue in Liver Samples
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The amount of interlobular connective tissue was measured in 4 μm thick FFPE tissue sections of liver samples only from the gilts. The tissue sections were stained with Goldner’s Masson trichrome stain to clearly depict fibrous connective tissues under a light microscope coupled with a digital camera. Using the software program NIS-Elements Basic Research (Nikon Instruments Inc., Tokyo, Japan), five consecutive microphotographs at HPF were made for each tissue section and represented the area of measurement. The microphotographs were then converted into a binary-colored output by marking pixels that belonged to either interlobular connective tissue or parenchyma, thus enabling automated detection of interlobular connective tissue. The amount of interlobular connective tissue was expressed as the area fraction of the corresponding pixels out of the total number of pixels in the area of measurement. When necessary, the automatically detected areas of interlobular connective tissue were corrected manually.
10
Immunofluorescence Analysis of NRF2 and XBP1S in Testis Tissue
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Testis tissues were prepared and deparaffinized as described above in the section “histology analysis”. Sections were immersed in a citrate solution and heated in a microwave oven for 15 min for antigen repair. Triton (0.1%, Keshi, China) and bovine serum albumin (0.5%, BSA, ZLI-9027, ZSGC-BIO) were applied for 15 min and 1 h, respectively. Primary antibodies (1:200) against NRF2 (Proteintech, 16396-1-AP) and XBP1S (Proteintech, 24868-1-AP) were used. Sections were incubated with primary antibodies at 4 °C for 12 h. A fluorescein-conjugated secondary goat anti-rabbit antibody (Proteintech, SA00009-2) was applied and incubated for 1 h at room temperature in the dark. Hoechst 33342 (Beyotime, C1022) was used as a nuclear counterstain (30 min incubation at room temperature). NIS Elements Basic Research (Nikon, USA) software was used to process the images.
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