Gene expression data from different wheat cultivars were used to analyze the gene expression profiles of the candidate region. Expression data were download from wheat-URGI website (https://wheat-urgi.versailles.inra.fr/Seq-Repository/Expression ). Then the transcriptomic information of candidate genes were exacted by a custom python script. Epidermal tissues were peeled off using tweezers under a stereomicroscope. Then, the cell layers were stained with safranin and mounted on glass slides (Matsunami Glass Ind., Japan). The tissue specimens were subjected to observation with a light microscope (BX50F Olympus Optical Co., Ltd, Japan).
Bx50f
Manufactured by Olympus
Sourced in Japan
The BX50F is a high-quality upright microscope designed for laboratory use. It features a sturdy frame and optical components that provide excellent image quality. The microscope is capable of various observation techniques, including brightfield, darkfield, and phase contrast.
Automatically generated - may contain errors
Lab products found in correlation
Glass slide, by Matsunami (1 mentions)
Ultravision lp detection system, by Thermo Fisher Scientific (1 mentions)
Microprobe manual staining system, by Thermo Fisher Scientific (1 mentions)
Image pro plus version 4, by Media Cybernetics (1 mentions)
Lsm 700, by Zeiss (1 mentions)
Eclipse 80i upright microscope, by Nikon (1 mentions)
Perfection v700 scanner, by Epson (1 mentions)
Micropublisher 3.3rtv, by Olympus (1 mentions)
Giemsa solution, by Kanto Chemical (1 mentions)
Hoechst 33258, by Merck Group (1 mentions)
Neutral buffered formalin solution, by Fujifilm (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Ba 3000, by Vector Laboratories (1 mentions)
Superfrost plus slide, by Thermo Fisher Scientific (1 mentions)
13 protocols using bx50f
1
Wheat Transcriptome Analysis by Tissue Isolation
2
Histological Evaluation of Skin Tissues
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Dorsal skin tissues (3 × 4 cm sizes) prepared from five mice per group were fixed in 4% paraformaldehyde, sectioned at 4 μm, deparaffinized, and stained with hematoxylin and eosin (H&E) to evaluate epidermal hyperplasia (thickness) or stained with toluidine blue to evaluate mast cell infiltration. For immunohistochemistry, staining experiments were conducted using the MicroProbe Manual Staining System (Fisher Scientific, Pittsburgh, PA, USA), some deparaffinized skin sections were immersed in 0.3% H2O2 to abolish endogenous peroxidase activity and incubated with polyclonal mouse anti-FLG (diluted 1:500) and anti-LOR (diluted 3 μg/mL) for 30 min at 38 °C. Skin sections were then treated with a secondary antibody and 3-amino-9-ethyl-carbacole as required by the UltraVision LP Detection System (Thermo Scientific, Waltham, MA, USA) and counterstained with hematoxylin for 30 s. All sections were photographed under a microscope (BX50F; Olympus Optical Co., Ltd.), and the images were analyzed using Image Pro Plus Version 4.5 software (Media Cybernetics Co., Silver Spring, MD, USA).
3
Microscopy Imaging and Analysis
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Immunohistochemistries were scored visually on an Olympus BX50F. CBAs and live hippocampal neurons were scored visually by two independent observers using a Nikon eclipse 80i upright microscope. Confocal images were acquired with a Zeiss LSM 700 using the 40× and 63× (oil) objectives. Images were processed using ImageJ.
4
Immunohistochemical Analysis of Ki67 Expression
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The tumor tissues exfoliated from the dead mice were paraffin-embedded, sectioned (4 μm), dewaxed with xylene, and hydrated with gradient alcohol. Then, the endogenous peroxidase was inactivated by blocking the sections with 3% H2O2 for 10 min, and the microwave repair was made with 0.01 mol/L sodium citrate buffer (pH = 6.0, 15 min). After that, the sections were blocked with 5% bovine serum albumin (BSA) for 20 min and incubated with the primary Anti-Ki67 antibody (1:1000, ab15580) at 4°C overnight. The next day, Goat Anti-Rabbit IgG (1:2500, ab6721) was added and incubated at RT for 20 min. After the sections were cleared with PBS, DAB was used for color development. The staining was observed under a microscope (BX50F; Olympus, Tokyo, Japan). The above antibodies were purchased from Abcam (MA, United States).
5
Root Architectural Traits Analysis
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Root architecture traits (length, average diameter, total root area) were measured using the WinRHIZO software (version 2012b) after scanning the roots using an Epson Perfection V700 scanner. Roots were separated in two groups based on their diameter according to Passot et al.39 (link): primary and crown roots (0.25 mm < diameters) and lateral roots (diameters < 0.25 mm).
Root hair length and density were measured on four plants per genotype using images of the root hair zone of three lateral roots per plant. Images were taken using an optical microscope (BX50F, Olympus) equipped with a digital camera (Micro Publisher 3.3 RTV). For each lateral root, the total number of root hairs was recorded over a distance of 0.5 mm using the Mesurim free software (http://acces.ens-lyon.fr/acces/logiciels/applications/mesurim/mesurim ) and the length of 10 randomly selected root hairs was measured using the ImageJ software.
Root hair length and density were measured on four plants per genotype using images of the root hair zone of three lateral roots per plant. Images were taken using an optical microscope (BX50F, Olympus) equipped with a digital camera (Micro Publisher 3.3 RTV). For each lateral root, the total number of root hairs was recorded over a distance of 0.5 mm using the Mesurim free software (
6
RBC Shape Evaluation by DIC Microscopy
7
Chromosome Number Estimation by Mitosis
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For chromosome number estimation, mitosis was observed using cells from root tips of regenerated plant #20, which were pretreated using 2 mM 8-hydroxyquinoline for 2–2.5 h at 20°C. After fixation, 1 mm of each root tip was cut off and macerated in enzyme solution consisting of 6.0% (w/v) Cellulase Onozuka RS (Yakult Pharmaceutical, Tokyo, Japan), 6.0% (w/v) Pectolyase Y-23 (Kyowa Chemical Products, Kagawa, Japan), and 75 mM KCl for 60 min at 37°C. The root tips were washed with a drop of distilled water for 5 min on a glass slide. To spread cells, each root tip was thoroughly squashed using a needle with 10 μl ethanol-acetic acid [3:1 (v/v)], and the slide was then flame-dried. The spread cells were stained for 30 min with Giemsa solution (Kanto Chemical Co., INC., Tokyo, Japan) diluted 30 times with Sorensen’s phosphate buffer (pH 6.8). After washing with distilled water, the number of chromosomes was counted under an optical microscope (Olympus BX-50 F, Olympus, Tokyo, Japan).
8
Apoptosis Detection by Hoechst Staining
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Since the chromatin condensation is a prominent hallmark of apoptosis, the apoptotic cells were recognized in this assay on the basis of nuclear condensation using Hoechst 33258 staining (Sigma, UK). To conduct DNA condensation assay, HGFs were plated at 1×105 cells per well in slide chambers (Nuns, Naperville, IL, USA). At the end of each treatment, treated and untreated (control) cells were fixed with cold methanol-acetone 1:1 (v/v) for 15 min, then washed and incubated with Hoechst 33258 staining (1 µg/mL) in the dark for 20 min. The condensation was evaluated under a fluorescence microscope (Olympus BX50F) equipped with a 3-CCD color video camera (Sony DXC) (Japan). All the experiments were performed in triplicate and repeated at three different times (n=3).
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Since the chromatin condensation is a prominent hallmark of apoptosis, the apoptotic cells were recognized in this assay on the basis of nuclear condensation using Hoechst 33258 staining (Sigma, UK). To conduct DNA condensation assay, HGFs were plated at 1×105 cells per well in slide chambers (Nuns, Naperville, IL, USA). At the end of each treatment, treated and untreated (control) cells were fixed with cold methanol-acetone 1:1 (v/v) for 15 min, then washed and incubated with Hoechst 33258 staining (1 µg/mL) in the dark for 20 min. The condensation was evaluated under a fluorescence microscope (Olympus BX50F) equipped with a 3-CCD color video camera (Sony DXC) (Japan). All the experiments were performed in triplicate and repeated at three different times (n=3).
9
Stomatal Characterization in Millets
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As leaves of millets have the amphistomatic type of stomatal distribution in both surfaces (39 ), stomatal counting was carried out on both the abaxial and adaxial surfaces by smearing with nail polish followed by shade drying (40 (link)). The smeared leaf was cut into 2.0–2.5 cm2 dimensions, and the layer of nail polish impression was gently removed. With the help of forceps, the same was placed on microscopic slides with a few drops of water and covered with a glass cover slip. Stomatal numbers on every sample surface were counted in three different microscopic fields of 10X and 40X magnifying lenses of a compound microscope (BX-50F, Olympus, Japan). Other related observations such as stomatal length, breadth, number of guard cells, and stomata in the particular microscopic field were recorded using user-friendly software.
10
Histopathological Evaluation of CNT Exposure
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Mice that survived 26 weeks after injection were euthanized by inhalation of isoflurane anesthesia. The brain, tongue, esophagus, trachea, thyroid, thymus, heart, lung, liver, kidney, spleen, stomach, small intestine, large intestine, testis, and epididymis were prepared according to the provision of the Central Institute for Experimental Animals (CIEA) (Kanagawa, Japan), an independent organization that promotes the development of quality humanized animals. Organs with macroscopically observed abnormalities were removed and fixed with 20% neutral buffered formalin solution (Wako Pure Chemical Industries, Osaka, Japan). Tissue specimens were also prepared under the CIEA guidelines. Each organ was cleaved according to the CIEA tissue assessment guidelines for rasH2 model mouse, and one hematoxylin and eosin stained tissue specimen was prepared per organ. Similarly, according to the provisions of CIEA, histological evaluation was performed with an optical microscope BX-50F (Olympus, Tokyo, Japan) to assess the presence of tumors. Moreover, the presence or absence of CNTs was confirmed with optical, fluorescence, and polarization microscopy using the same tissue sample.
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