Sheep ( ALC and MF) and mouse skin tissues were fixed in 4% paraformaldehyde formalin in PBS at 4 °C overnight, embedded in paraffin, sectioned at 5 μm, and stained with a hematoxylin and eosin staining kit (H&E). The following antibodies were used for immunostaining: anti-Ki67 (Abcam, ab15580), anti-Caspase 3 (Abcam, ab184787), anti-AKT1 + AKT2 + AKT3 (Abcam, ab179463); phospho-PI3-kinase p85-alpha/gamma (Abmart, T40116F); mTOR antibody (Abmart, T55306); HRP-labeled goat anti-rabbit IgG(H+L) (Beyotime, A0208), and HRP-labeled goat anti-mouse IgG(H+L) (Beyotime, A0216). The signal was detected using the DAB Horseradish Peroxidase Color Development Kit (Beyotime, P0202), and the sections were stained with hematoxylin. Photographs were taken using a microscope (ECHO, RVL-100-G, USA).
Rvl 100 g
Manufactured by Echo Medical Systems
Sourced in United States
The RVL-100-G is a laboratory equipment product from Echo Medical Systems. It is designed for use in various scientific and medical research applications. The core function of the RVL-100-G is to perform precise measurements and analyses, though the specific intended use is not provided in this factual and unbiased description.
Automatically generated - may contain errors
Lab products found in correlation
Cm1900, by Leica (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Dab horseradish peroxidase color development kit, by Beyotime (1 mentions)
Hrp labeled goat anti mouse igg h l, by Beyotime (1 mentions)
Anti akt1 akt2 akt3, by Abcam (1 mentions)
Mtor antibody, by Abmart (1 mentions)
Hrp labeled goat anti rabbit igg h l, by Beyotime (1 mentions)
T55306, by Abmart (1 mentions)
Calcein am, by Solarbio (1 mentions)
M5904, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
α mem, by Thermo Fisher Scientific (1 mentions)
Methocult gf m3434, by STEMCELL (1 mentions)
Pk90017s, by Abmart (1 mentions)
Cy3 labeled goat anti rabbit igg, by Beyotime (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Tcs sp8, by Leica (1 mentions)
C1090, by Beyotime (1 mentions)
Enspire, by PerkinElmer (1 mentions)
20 protocols using rvl 100 g
1
Histological Analysis of Skin Tissues
2
Immunofluorescence Staining of Skin Tissue
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Frozen sections of skin tissue up to 6 µm thick were prepared and fixed in pre-chilled acetone for 10 min, then washed three times with PBS for 10 min each and incubated for 30 min at room temperature with 0.5% Trition100 (50 µL of trition100 in 100 ml of PBS). Trition incubation was not required for immunofluorescence experiments with mTOR antibody (Abmart, T55306) in this study. PBS was washed three times for 10 min each and blocked with 1% BSA for 1.5 h. The antibodies (anti-AKT1 + AKT2 + AKT3:Abcam, ab179463, phospho-PI3-kinase p85-alpha/gamma:Abmart, T40116F, mTOR antibody:Abmart, T55306) were diluted in BSA and incubated overnight in a wet box at 4 °C. The antibodies were recovered and the sections were washed 3 times with PBS for 10 min, added with a fluorescent secondary antibody corresponding to the primary antibody, incubated for 1 h at room temperature in a wet box, washed 3 times with PBS, added with DAPI and photographed under a microscope (ECHO, RVL-100-G, USA).
3
Quantifying Live/Dead Cell Ratio
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According to the standard of 1×105 cells per well, 500 μL cell suspension was put into 48-well plate. After the cells adhered to the wall, the original cell culture medium was replaced with the extraction solution. After culturing for 3 days, replace the normal cell culture medium with 1.5 μL propidium iodide and 1 μL calcein-AM (Solarbio, China) in PBS solution, and incubate for another 30 min. After the sample was gently washed with PBS, fluorescence was excited by 490 nm wavelength under inverted fluorescence microscope (ECHO RVL-100-G, United States), and the distribution of living dead cells was observed.
4
Morphological Characterization of U. prolifera
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Samples of U. prolifera were photographed and observed for morphological characteristics of thalli such as the color, number, and shape of branches. Then, the thalli were sheared into 2‐mm slices to observe cellular details using a light microscope (RVL‐100‐G, ECHO, USA). The main branches of samples from SH and NH were cut into 400‐mm lengths to calculate the SA:VOL. The surface area was calculated using ImageJ (National Institutes of Health, USA) and HTPheno (IPK, Germany; Macinnis‐Ng et al., 2005 (link); Ralph et al., 1998 (link)). Volume was calculated by a volumetric cylinder using water.
5
Quantifying Germ and Vegetative Cells
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Thalli were cleaned with a brush, washed with sterile seawater, and placed on glass slides. The cells were observed using a light microscope (RVL‐100‐G, ECHO). The number of germ cells and the number of vegetative cells were counted to calculate RA (%) (Wang et al., 2019 (link)).
Here, Ng is the number of germ cells and Nv is the number of vegetative cells.
Here, Ng is the number of germ cells and Nv is the number of vegetative cells.
6
Quantifying BoHV-4 Infection and Replication
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To investigate virus infection and replication, MDBK cell monolayers grew on coverslips were infected with BoHV-4 at 0.1, 0.25, 0.5, 1, and 2.5 MOI. At 72 h post infection (hpi), the cells were fixed with 4% formaldehyde at 37 °C for 40 min. Then, the cells were washed with PBS buffer and blocked with PBS containing 5% non-fat milk at 37 °C for 45 min. After three washes with PBS buffer, the cells were incubated with BoHV-4 polysera (1:100 dilution) as primary antibody for 40 min. The cells were washed with PBS and incubated with anti-mouse IgG antibody as secondary antibody for 40 min. Finally, the cells were washed with PBS and stained with 4′,6-diamidino-2-phenylindole (DAPI). Images were captured under an ECHO fluorescence microscope (RVL-100-g, USA). Notably, BoHV-4 polysera was prepared from mice challenged with BoHV-4.
7
Effects of Temperature on Giant Unilamellar Vesicles
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GUVs solution were incubated in the dark at temperatures of 4 °C, 25 °C, 37 °C, and 45 °C. Samples were taken on day 0, day 1, day 3, day 5, and day 7 of incubation, respectively. Before sampling, the homogenous solution was thoroughly mixed. Then, 10 μL of the GUVs solution was dropped into the glass bottom cell culture dish (801001, NEST, China), sealed with mineral oil (M5904, Sigma, USA), and allowed to settle for 3 h in the dark. The samples were viewed using an inverted fluorescence microscope (RVL-100-G, ECHO, San Diego, CA, USA) or a confocal laser scanning microscope (CLSM, A1R; Nikon, Tokyo, Japan), and the center of the GUVs solution and eight directions around it were photographed and recorded. Experiments were repeated three or more times and used for subsequent data analysis. Individual GUVs or aggregates were observed with a high-power lens and the Z-axis layer sweep recombination technique of CLSM (thickness is 0.5 μm). The excitation and emission wavelengths of DiO are 488 nm and 500–520 nm, and the excitation and emission wavelengths of DiI are 551 nm and 569 nm, respectively.
8
Immunofluorescence Imaging with Secondary Antibody
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After the biotin-labeled secondary antibody was replaced with goat anti-rabbit IgG (0.125*10-2mg/ml, bs-0259G-AF488, Bioss), and incubated at 4°C for 1 hr in a cassette. The rest of the programs were the same at the operating procedures of IHC. In the end, immunofluorescence images were collected by a microscope (RVL100-G, ECHO, USA).
9
Hematopoietic Colony-Forming Assay for Embryonic Tissues
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Hematopoietic colony-forming assay was designed to assess the hematopoietic potential and was performed according to the protocols described by Nakano et al. (2013 (link)). Briefly, the embryonic tissues including YS, caudal half, head, and heart regions were dissected and precultured on OP9 stromal cells for 4 d at 37°C in 24-well plates containing 1 ml α-MEM (Gibco) containing 20% fetal bovine serum (Gibco) and 1% penicillin/streptomycin supplemented with stem cell factor (50 ng/ml), thrombopoietin (5 ng/ml), IL-3 (IL-3, 5 ng/ml), IL-6 (IL-6, 5 ng/ml), and Flt-3 ligand (Flt-3L, 10 ng/ml). After 4 d, the tissues were transferred from 24-well plates into 1.5 ml tubes and dissociated mechanically by pipetting, and the stromal cells were removed through filtering. The cells were then transferred into methylcellulose supplemented with IL-3, IL-6, stem cell factor, and erythropoietin (MethoCult GF M3434; Stem Cell Technologies) in 1:10 ratio, followed by mixing well and plating with 400 μl medium at the bottom of the 24-well plates. The cells were cultured at 37°C for 10 d. The hematopoietic colonies were analyzed with microscope (ECHO/RVL-100-G) according to the manufacturer’s instruction.
10
Erythrocyte Morphology in bHb-HES Presence
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The morphology
of RBCs in the presence of bHb-HES130 and bHb-HES200 was measured
using an inverted optical microscope (RVL-100-G, ECHO, USA). bHb,
bHb-HES130, and bHb-HES200 (0.3 mg bHb) were incubated with BRCs at
0.6% Hct for 30 min. The specimens were prepared by dropping the mixtures
(20 μL) onto the slide followed by covering with the cover glass.
The specimens were observed under the inverted optical microscope.
The magnification was adjusted to 40× to take a photo.
of RBCs in the presence of bHb-HES130 and bHb-HES200 was measured
using an inverted optical microscope (RVL-100-G, ECHO, USA). bHb,
bHb-HES130, and bHb-HES200 (0.3 mg bHb) were incubated with BRCs at
0.6% Hct for 30 min. The specimens were prepared by dropping the mixtures
(20 μL) onto the slide followed by covering with the cover glass.
The specimens were observed under the inverted optical microscope.
The magnification was adjusted to 40× to take a photo.
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