After the reperfusion, fresh heart biopsies were fixed in 4% paraformaldehyde overnight at 4°C and embedded in paraffin. Sections into 5-μm slices were stained with hematoxylin-eosin (H&E) or Masson’s trichrome for assessment of fibrosis. Tissues for immunofluorescence were submerged in liquid nitrogen and then embedded in optimal cutting temperature (OCT) solution (Sakura Finetek, USA) on dry ice to be frozen completely. Cardiomyocyte hypertrophy was examined in the peri-infarct zone. Myocyte cross-sectional areas were measured using Image J software (National Institutes of Health) in frozen sections stained with 5 μg/ml wheat germ agglutinin (WGA-Alexa Fluor® 488 conjugate, Invitrogen, USA). Five parts were chosen in the WGA images (200X) including left top, right top, middle, left bottom, and right bottom, and six cells were analyzed for each part. In other experiments, the hearts were excised for Masson staining to evaluate the cardiac remodeling. For inflammatory cell infiltration and PDGFR protein expression, immunofluorescence staining with anti-CD45 (Cell Signaling Technology, USA) and anti-PDGFRα (Cell Signaling Technology, USA) in frozen sections was conducted. Then the number of CD45+ cells/field were quantified by Image J software (National Institutes of Health, USA).
Wga alexa fluor 488 conjugate
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Germany, France
The WGA) Alexa Fluor 488 conjugate is a fluorescent labeling reagent used for the detection and visualization of glycoproteins in biological samples. It consists of wheat germ agglutinin (WGA) covalently conjugated to the Alexa Fluor 488 dye. The Alexa Fluor 488 dye provides green fluorescence upon excitation, allowing for the localization and quantification of glycoproteins in various applications such as flow cytometry, fluorescence microscopy, and protein analysis.
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Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (6 mentions)
Lsm 880, by Zeiss (4 mentions)
Lsm 710, by Zeiss (4 mentions)
Dmi600b, by Leica (3 mentions)
Prolong gold, by Thermo Fisher Scientific (2 mentions)
8 well chamber slide, by Ibidi (2 mentions)
Zen blue software, by Zeiss (2 mentions)
Vt1200, by Leica (2 mentions)
Propidium iodide, by Merck Group (2 mentions)
Vt1200s vibratome, by Leica (2 mentions)
Masson ht15 1kt, by Merck Group (2 mentions)
Propidium iodide, by Thermo Fisher Scientific (2 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Oct solution, by Sakura Finetek (1 mentions)
Anti cd45, by Cell Signaling Technology (1 mentions)
Anti pdgfrα, by Cell Signaling Technology (1 mentions)
Isoproterenol, by Merck Group (1 mentions)
Alzet model 2001, by Durect (1 mentions)
Picrosirius red stain kit, by Polysciences (1 mentions)
43 protocols using wga alexa fluor 488 conjugate
1
Cardiac Tissue Analysis Post-Infarction
2
Isoproterenol-Induced Myocardial Necrosis
3
Quantifying Cardiac Fibrosis and Cardiomyocyte Size
4
Immunofluorescence and Autophagy Analysis
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Differentiated EBs and monolayer cultures were fixed with 4% paraformaldehyde (PFA) and permeabilized with 0.1% Triton X-100 at RT. The following primary antibodies were used: MF20 (1 : 50, Developmental Studies Hybridoma Bank); βIIITubulin (1 : 400, Sigma-Aldrich). After washing in 0.5% Tween-1 × PBS, cells were incubated with secondary antibodies (1 : 200, Alexa Fluor, Molecular Probes Inc., Life Technologies).
For autophagy detection, cytospin samples (see above) were stained with LysoTracker Red DND-99 dye. Briefly, cells (50 000 cells/spot) were washed with PBS 1 × , stained with LysoTracker Red DND-99 dye (Molecular Probes Inc.) for 1 min at room temperature. Cells were gently washed and fixed in 4% PFA for 30 min, washed three times with PBS, and stained with membrane stain WGA-Alexa Fluor 488 Conjugate (Invitrogen) following the manufacturer's instructions. Cell nuclei were counterstained with Hoechst 33342 (Invitrogen). Images were obtained using the DMI6000B microscope and the DFC 350FX B/W digital camera (Leica, Solms, Germany). Leica FW4000 and AF6000 software were used for image acquisition/elaboration. Confocal images were acquired at × 63 magnification on a LSM710 confocal fluorescence microscope (Carl Zeiss Inc., Jena, Germany) using the ZEN 2008 software (Carl Zeiss Inc.).
For autophagy detection, cytospin samples (see above) were stained with LysoTracker Red DND-99 dye. Briefly, cells (50 000 cells/spot) were washed with PBS 1 × , stained with LysoTracker Red DND-99 dye (Molecular Probes Inc.) for 1 min at room temperature. Cells were gently washed and fixed in 4% PFA for 30 min, washed three times with PBS, and stained with membrane stain WGA-Alexa Fluor 488 Conjugate (Invitrogen) following the manufacturer's instructions. Cell nuclei were counterstained with Hoechst 33342 (Invitrogen). Images were obtained using the DMI6000B microscope and the DFC 350FX B/W digital camera (Leica, Solms, Germany). Leica FW4000 and AF6000 software were used for image acquisition/elaboration. Confocal images were acquired at × 63 magnification on a LSM710 confocal fluorescence microscope (Carl Zeiss Inc., Jena, Germany) using the ZEN 2008 software (Carl Zeiss Inc.).
5
Mucus Secretion and Bacterial Interactions in Cocultures
6
In Vitro Binding Assay of FSH-Rh760 and Rh760
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To test the in vitro binding capacity of FSH-Rh760 and Rh760, A2780, IGROV1 and SKOV3 ovarian cancer cells were seeded in 8-well chamber slides (Ibidi) and grown to 50 %–60 % confluence. Cells were incubated with 10 μmol/L FSH-Rh760 or Rh760 for 90 min at 37 °C. After incubation, the cells were washed with PBS buffer, fixed in 4 % paraformaldehyde, labeled with WGA Alexa Fluor 488 conjugate (Invitrogen), and then sealed with mounting medium containing DAPI. The xenografts and major organs from the mouse model were sectioned into frozen slices of 7 μm thickness. Slides were fixed and mounted. These samples were analyzed using TCS SP5 confocal fluorescence microscopy (Leica).
7
Visualizing Yeast Cell Maturity and Mitochondrial Potential
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The maturity and size of cells were determined based on bud scars and MMP. Briefly, yeast cells were harvested on day 14 of the CLS analysis and then simultaneously stained in 500 µL of DPBS containing 50 μg/mL wheat germ agglutinin (WGA) Alexa Fluor 488 conjugate (Invitrogen, Waltham, MA, USA) for 45 min and 180 nM TMRE for 30 min at 30 °C with shaking (350 rpm). Z-stack live-cell imaging was performed using HC PL APO 63×/1.4 oil-immersion objective with 2 × digital zoom. Images were sequentially scanned at an excitation/emission wavelength of 488/500–560 nm for green fluorescence (bud scars) and 543/570–610 nm for red fluorescence (MMP). The analysis was performed based on the ROI area. Z-projection was created using the “max” intensity option of LAS X 3.3.3 software, and then, the ROIs were selected to measure the red and green fluorescence intensity of each yeast cell.
8
Multicolor Imaging of Mitochondria and Cell Membrane
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Cells were seeded in duplicates with a density of 2 × 104 cells in 50 µL per well on an 8-well Nunc™ Lab-Tek™ Chambered Coverglass dish (Thermo Fisher Scientific, Rochester, NY, USA) and incubated for 24 h together with the compounds. Immediately before analysis, 5 µL of 20 µM HEPES buffer (Biochrom GmbH, Berlin, Germany) was added to each cavity. Subsequently, 2 µL of reduced MitoTracker®Red-H2XROS (Invitrogen by Thermo Fisher Scientific, Eugene, OR, USA) and 2 µL of Wheat Germ Agglutinin (WGA)-Alexa Fluor™ 488 conjugate (Invitrogen by Thermo Fisher Scientific, Eugene, OR, USA) were added and incubated for 15 min. Hoechst 33342 (Invitrogen by Thermo Fisher Scientific, Eugene, OR, USA) was added 5 min before the cells were analyzed by confocal microscopy utilizing an inverted microscope (Zeiss Axio Observer Z1, Zeiss, Oberkochen, Germany) in arrangement with a spinning disk confocal system (UltraVIEW VoX, PerkinElmer, Waltham, MA, USA). All the images were generated using a 40× water immersion objective (Zeiss, Vienna, Austria).
9
Visualizing EV-mediated Nucleic Acid Transfer
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Thirty microliters of the HEK-CD63-GFP TSU and PSU sEVs (F1–F5) containing EV-DNA-EdU was incubated with HeLa cells seeded on a 24-well plate containing sterile 12 mm microscopic coverslips at 37 °C for 48 h. In parallel, MV4−11 sEVs containing EdU were added to mouse mesenchymal stromal cells (OP9). Fixation and permeabilization were performed as explained before. If cell membrane staining was desired, cells were treated with wheat germ agglutinin (WGA) Alexa Fluor™ 488 conjugate (W11261, Invitrogen) for 10 min well after fixation, before permeabilization. An EdU click-it reaction was carried out using the Click-iT™ EdU Alexa Fluor 647 Imaging Kit (C10640, ThermoFisher Scientific) following the manufacturer’s instructions. Images were acquired by confocal microscopy in the corresponding channels (blue—DAPI, green—CD63+ EVs, and red—EdU). Using ImageJ, the red fluorescence signal corresponding to EV-DNA was quantified.
10
Fluorescent Imaging of GnRH Receptor Binding
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The cells were placed in an 8-well chamber slide (Ibidi) and grown to ~60% confluence. A2780, CAOV-3, ES-2, and HeyA8 cells were incubated with 100 μmol/L GnRHa-FITC, and A2780, OSE, and H1299 cells were incubated with 20 μmol/L GnRHa-ICG or ICG (Sigma-Aldrich) for 60 min at 37°C. After incubation, the cells were washed with PBS, fixed in 4% paraformaldehyde, labeled with WGA Alexa Fluor 594 conjugate or WGA Alexa Fluor 488 conjugate (Invitrogen), and mounted with DAPI. These samples were analyzed using TCS SP5 confocal microscopy (Leica). For quantitative analysis, the mean fluorescence intensity was calculated using ImageJ software (version 1.50 g).
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