Similar anatomical areas among animals were selected for the histological analysis. Four sections for each animal were analyzed. Colocalization of aromatase and GnRH was studied by immunofluorescence. Microscope images were captured using a Nikon C1 Plus Laser microscope (Nikon Inverted Research Microscope Eclipse Ti, Nikon Corp., Tokyo, Japan) and analyzed with the EZ‐C1 software (EZ‐C1 software v3.9, Nikon Ltd., London, United Kingdom). Around 20 cells per section were counted at POA and the percentage of cells showing aromatase and GnRH colocalization was quantified. Adobe Photoshop software (Adobe Photoshop CS5, Adobe Systems Inc., Ottawa, Ontario, Canada) was used for digital manipulation of brightness and contrast when preparing the shown images.
Inverted research microscope eclipse ti
Manufactured by Nikon
Sourced in Japan, China
The Inverted Research Microscope Eclipse Ti is a high-performance optical microscope designed for advanced research applications. It features a versatile inverted configuration, allowing for the observation of samples from below. The Eclipse Ti provides excellent optical performance and a range of advanced functionality to support various imaging techniques, such as phase contrast, fluorescence, and DIC microscopy.
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Lab products found in correlation
Numerical aperture objective, by Nikon (5 mentions)
Crystal violet, by Beyotime (4 mentions)
Live dead baclight fluorescence stain, by Thermo Fisher Scientific (2 mentions)
Mc170 hd, by Nikon (2 mentions)
Ez c1, by Nikon (1 mentions)
Matrigel solution, by BD (1 mentions)
5 ethynyl 2 deoxyuridine edu assay kit, by RiboBio (1 mentions)
Anti p fak, by Cell Signaling Technology (1 mentions)
Anti vinculin, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Dapi fluoromount g, by Southern Biotech (1 mentions)
Hoechst, by Merck Group (1 mentions)
Catalase, by Merck Group (1 mentions)
Dulbecco s pbs, by Merck Group (1 mentions)
As 55043a, by AnaSpec (1 mentions)
24 protocols using inverted research microscope eclipse ti
1
Colocalization of Aromatase and GnRH
2
Intracellular Reactive Oxygen Sensing
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2’,7’-Dichlorodihydrofluorescein diacetate (DCFH-DA) and APF dyes were utilized to sense the intracellular H2O2 and ‧OH radicals, respectively. The cancer cells were seeded in 96-well plates (8000 HepG2-Red-FLuc and A549 cells/per well) and incubated for 24 h, followed by the treatments of DCFH-DA or APF dyes with the CFPB and CFPB@Lipo nanoframes (28 ppm in cobalt ion concentration) for another 24 h. The cells were treated by the medium without the nanomaterials as the control group. And then, the cells were gently rinsed twice for further observation by laser scanning confocal microscope (Nikon inverted research microscope ECLIPSE Ti). The dyes of propidium iodide (PI) and Calein-AM were used to stain the dead and live cells, respectively. The cancer cells were seeded in 96-well plates (8000 HepG2-Red-FLuc and A549 cells/per well) and incubated for 24 h, followed by the treatments of medium alone, CFPB or CFPB@Lipo nanoframes (28 ppm in cobalt ion concentration) for another 24 h. After treatments, the cells were gently rinsed twice and further stained by PI and Calein-AM following standard process. The distribution of dead and live cells was observed by laser scanning confocal microscope.
3
Transwell Assay for Cell Migration and Invasion
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Transwell assay was conducted for the detection of cell migration and invasion. After 48 h of transfection, 1 × 105 cells in 200 μl of serum-free medium were placed in the upper chamber (8.0 μm pore size; Corning, USA; Catalog number 3422) with a porous membrane with Matrigel solution (BD, USA) for invasion assay, while the lower chamber was inserted into a 12-well filled with 600 μl medium added with 10% FBS. After 24 h of incubation at 37 °C, noninvasive cells were removed from the upper surface of the membrane with cotton swabs, and invasive cells on the lower membrane surface were fixed with 4% formaldehyde and stained with 0.1% crystal violet (Beyotime, China). Five random 200 × visual fields per well were photographed and calculated under a Nikon Inverted Research Microscope Eclipse Ti microscope. Cell migration assay was simultaneously conducted as above, except for the chambers without Matrigel.
4
Intestinal Organoids Cytokine Response
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Intestinal organoids cultured alone, treated with TNF-α, and co-cultured with RAW 264.7 macrophages were treated with 5 μM Forskolin and directly analyzed by live cell microscopy (Nikon Inverted Research Microscope ECLIPSE Ti, Amstelveen, The Netherlands).
5
Pulsed Electric Field Effects on TPC-1 Cell Proliferation
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The proliferative ability of pulsed TPC-1 cells was examined using the 5-ethy-nyl-2′-deoxyuridine (EdU) assay kit (Ribobio). As a thymine nucleoside analogue, EdU is able to replace thymine during cell proliferation by infiltrating into replicating DNA molecules. The cell proliferation rate can then be measured by double labeling the nucleus in combination with nuclear markers (e.g., Hoechest 33342). TPC-1 cells treated with pulsed electric field were cultured in medium-containing EDU (final concentration of 10 μm) at 37°C for 2 h, and then the cells were washed with PBS 1 ~ 2 times, each time for 5 min. After adding 1 mL 4% paraformaldehyde, the cells were fixed at room temperature for 15 min. After removing the fixed solution, the samples were washed with PBS and then incubated with PBS containing 0.5% Triton X-100 for 10 minutes. Hoechst 33342 was stained for 30 min. After washing, the staining results were observed by inverted fluorescence microscope (Nikon Inverted Research Microscope ECLIPSE Ti). For each staining result, five random fields were imaged at ×20 magnification. The image was analyzed with Image-Pro Plus software. EdU incorporation rate was expressed as the ratio of the number of EdU-positive cells to the total number of cells in each field.
6
Quantitative Immunofluorescence Analysis
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Immunofluorescence images were carried out using a microscope (Inverted Research Microscope Eclipse Ti, Nikon) equipped with an epi-fluorescence illuminator or a 488 nm laser for Total Internal Reflection Fluorescence (TIRF) application. For sample preparation, cells were fixed in 4 % paraformaldehyde for 20 min, washed in PBS, permeabilized with 0.5 % PBS-Tween for 10 min and 0.1 % PBS-Tween for 5 min (three times). Subsequently, cells were blocked in 1 % BSA in 0.1 % PBS-TWEEN for 60 min. Antigen recognition was performed by incubating primary antibody in a humidified chamber for different times (Phalloidin, Invitrogen, A12381; anti-Vinculin, Invitrogen, 42H89L44; anti-pFAK, Cell signaling, 3283S) and with antimouse/rabbit Alexa Fluor 488-594 (Invitrogen, A11008, A11005) as secondary antibody in a humidified chamber for 60 min. Nuclei were stained with DAPI (Sigma-Aldrich). Images were analyzed by using ImageJ®. For the image processing, the F-actin total intensity fluorescence/cell area, nuclear area, FA area and number (by considering an analyze particle threshold of 0.25 μm 2 ) were normalized to the number of cells present in each image.
7
Colony Formation Quantification Protocol
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A total of 500 cells per well were plated into 6-well plates. After the cells were grown for about 10 days, cells were xed with 4% paraformaldehyde and stained with crystal violet (Beyotime, China) for 30 min, and colonies were photographed under a Nikon Inverted Research Microscope Eclipse Ti microscope and quanti ed with imageJ4.
8
Colony Formation Quantification Protocol
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A total of 500 cells per well were plated into 6-well plates. After the cells were grown for about 10 days, cells were xed with 4% paraformaldehyde and stained with crystal violet (Beyotime, China) for 30 min, and colonies were photographed under a Nikon Inverted Research Microscope Eclipse Ti microscope and quanti ed with imageJ4.
9
Colony Formation Quantification Protocol
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A total of 500 cells per well were plated into 6-well plates. After the cells were grown for about 10 days, cells were xed with 4% paraformaldehyde and stained with crystal violet (Beyotime, China) for 30 min, and colonies were photographed under a Nikon Inverted Research Microscope Eclipse Ti microscope and quanti ed with imageJ4.
10
Immunofluorescence Staining of Transfected Cells
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Prior to cell seeding, coverslips were put on the six-well plates. Cells were cultured in the six-well plates and transfected with PLK1 or KPNB1 siRNA. Following transfection, cells were incubated at 37°C with 5% CO2. Cells were washed with phosphate-buffered saline (PBS) three times and fixed with 4% paraformaldehyde in PBS. Cells were rinsed with PBS three times, followed by incubation with 0.2% Triton X-100 in PBS for 5 min at room temperature. Cells were washed with PBS three times and blocked in PBS containing 1% BSA, followed by incubation with the appropriate primary antibody overnight at 4°C. Cells were washed with PBS five times, followed by incubation with the appropriate secondary antibody in the dark for 50 min at room temperature. Cells were then washed with PBS five times. The cells were mounted with Dapi-Fluoromount-G (Southern Biotechnology Associates Inc., Birmingham, AL, USA). Images of the cells were taken using an ECLIPSE Ti inverted research microscope (Nikon, Tokyo, Japan) and analyzed using ImageJ software 18 (link).
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