Seven-day-old seedlings were incubated with 50 µM MDC (Monodansylcadaverine, Sigma) in PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4) for 15 min. Then the seedlings were washed with PBS buffer for two times and imaged by Leica DM IRB epifluorescent microscope with UV filter. Images were recorded by Leica DFC350 FX camera.
Dmirb epi fluorescent microscope
Manufactured by Leica
Sourced in Germany
The DMIRB epi-fluorescent microscope is a laboratory equipment designed for fluorescence imaging. It features a compact and modular design, enabling versatile configurations for various applications. The microscope utilizes epi-fluorescence illumination, which provides efficient excitation and detection of fluorescent samples.
Automatically generated - may contain errors
Lab products found in correlation
Dfc350 fx camera, by Leica (3 mentions)
Click it edu alexa fluor 488 imaging kit, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole dihydrochloride dapi, by Thermo Fisher Scientific (2 mentions)
Sma primary antibody, by Merck Group (2 mentions)
Alexa fluor 568 conjugated mouse secondary antibody, by Thermo Fisher Scientific (2 mentions)
Sp8 confocal microscope, by Leica (2 mentions)
Click it edu imaging kit, by Thermo Fisher Scientific (2 mentions)
Monodansylcadaverine mdc, by Merck Group (1 mentions)
8 protocols using dmirb epi fluorescent microscope
1
Visualizing Autophagy in Plant Seedlings
2
EdU Incorporation Assay in Arabidopsis
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Edu (5-ethynyl-2′-deoxyuridine) staining was performed using Click-iT® EdU AlexaFluor® 488 Imaging Kit (Invitrogen). Seven-day-old seedlings grown on AT medium (pH 5.8, 0.6% agar) supplemented with 30 mM Glc were transferred to AT medium supplemented with 30 mM Glc and 5 µM AZD-8055 or 0.5 µM chlorsulfuron for 2 h. Then 5 µl 1 μM EdU in liquid AT medium was added directly on the root tip. The root tips were incubated with EdU for 30 min in the climate chamber. Then the seedlings were fixed in 100 μl fixation/permeabilization reagent (4% formaldehyde, 0.1% Triton X-100 in 1× PBS) for 30 min. After fixation, seedlings were incubated in the dark with 100 μl Click-iT reaction cocktail (prepared according to the manufacturer’s protocol, Click-iT® EdU AlexaFluor® 488 Imaging Kit, Invitrogen). The seedlings were washed with PBS buffer for two times. All the samples were analyzed by Leica DM IRB epifluorescent microscope with FITC/GFP filter (AlexaFluor 488: excitation 495 nm; emission 519 nm). Images were recorded by Leica DFC350 FX camera.
3
Immunofluorescence Analysis of Smooth Muscle Actin
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Smooth muscle actin (SMA) expression in explanted cells was analyzed by immunofluorescence. SMA primary antibody (Sigma A2547, dilution 1:500) was diluted in 2% bovine serum albumin (BSA; EMD Chemicals, San Diego, CA) in PBS. Primary antibodies were detected using 1:500 Alexa Fluor® 568 conjugated mouse secondary antibody (Life Technologies A11004, Waltham, MA) and nuclei were detected with 1:10,000 4’,6-diamidino-2-phenylindole,dihydrochloride (DAPI; Life Technologies D1306) diluted in 2% BSA in PBS. Imaging was performed on a Leica SP8 confocal microscope (Wetzlar, Germany) or a Leica DMIRB epi-fluorescent microscope (Wetzlar, Germany).
4
Jagged1-Induced Cell Proliferation Analysis
5
Jagged1-Induced Cell Proliferation Analysis
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Proliferation of explanted cells was analyzed using the Click-iT® EdU imaging kit (Invitrogen C10337, Waltham, MA). Cells were plated on Jagged1-Fc and Fc control coated 24-well plates, allowed to incubate for 24 hours, and then treated with 10 µM 5-ethynyl-2´-deoxyuridine (EdU) diluted in SmGM2 for an additional 24 hours. Cells were then fixed using 3.7% formaldehyde and permeabilized in 0.5% Triton® X-100. Click-iT® EdU detection was performed as well as nuclear staining with 4’,6-diamidino-2-phenylindole (DAPI). At least 3 wells per treatment were each imaged in 7 zones on a Leica DMIRB epi-fluorescent microscope (Wetzlar, Germany). Thresholding, watersheding, and particle analysis was completed in ImageJ (NIH Bethesda, MD) for the DAPI channel and the EdU channel for each image so the ratio of EdU positive cells to total cells could be calculated as a percentage.
6
Plasmid Transfection via DEP Trapping
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For plasmid transfection, cells grown in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% fetal bovine serum and penicillin/streptomycin were trypsinized and suspended in 7% sucrose solution at a density of 3 × 106 cells/mL. 13.47 μg of plasmid vector tagged with RFP and 10 μL of 4 mM Calcein AM was added to the cell solution. The mixture was introduced into the system under a 20 nL/min constant flow rate and plasmid transfection was carried out in the following sequence; (1) DEP trapping field of 4VPP was left on for 2 minutes to simultaneously immobilize and electroporate cells at trap locations. Visual confirmation was obtained via concurrent imaging with a Leica DMIRB epifluorescent microscope. (2) Flow rate was increased to 5 μL/min and cells were collected into 200 μL of DMEM with 10% fetal bovine serum and 20 mM HEPES with penicillin/streptomycin in a 1.5 mL microcentrifuge tube. The entire contents containing ≈10,000 cells was transferred to a 96-well plate for culture in a 37 ºC incubator. The process was repeated 3x for each of the test and control conditions. Cells were cultured overnight and imaging of a 440 μm × 330 μm region of the cultured well was completed to determine if transfection had occurred. A detailed description of the plasmid design is provided Supplementary Section S7 .
7
Quantifying Cell Proliferation in Roots
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Edu (5-ethynyl-2′-deoxyuridine) staining was performed using a Click-iT EdU Alexa Fluor 488 Imaging Kit (Invitrogen). Two-week-old seedlings were grown on AT medium (pH 5.8, 0.6% Agar) containing 500 µM sulfate (+S) or 0 µM sulfate (-S). Seedlings grown on -S medium were transferred to medium supplemented with 1% sucrose (-S+Suc) for 24 h. Five milliliter 1 μM EdU in liquid AT medium was added directly to the root tip. The root tips were incubated with EdU for 30 min in a climate chamber. The seedlings were fixed in 100 μL Fixation/Permeabilization reagent (4% formaldehyde, 0.1% Triton X-100 in 1× PBS) for 30 min. After fixation, the seedlings were incubated in the dark with 100 μL Click-iT reaction cocktail (prepared according to the manufacturer's protocol, Click-iT EdU Alexa Fluor 488 Imaging Kit, Invitrogen). The seedlings were washed twice with PBS buffer. All samples were analyzed under a Leica DM IRB epifluorescent microscope with FITC/GFP filter (AlexaFluor 488: excitation 495 nm; emission 519 nm). Images were recorded with a Leica DFC350 FX camera.
8
Immunofluorescence Analysis of Smooth Muscle Actin
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