Cell death was assessed by staining the cells with the vital dye Hoechst 33342, which evidences nuclei and allows for the detecting of chromatin condensation and fragmentation. For these experiments, 7 × 103 cells/well were seeded in a 96-well plate, incubated with the compounds for the established times and then stained with Hoechst (2.5 µg/mL medium) for 30 min. After washings with PBS, cells were visualised using an inverted Leica fluorescent microscope (Leica Microsystems, Wetzlar, Germany) endowed with a 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI) filter. Leica Q Fluoro Software (Leica Microsystems, Wetzlar, Germany) was used for image acquisition. Annexin V apoptosis detection assay was used to evidence early apoptotic cells. Briefly, 3 × 105 cells were seeded in 6 cm diameter Petri dishes, allowed to adhere overnight and then treated with the compounds for 24 h. Cells were then harvested, washed twice in PBS, and 105 were incubated for 15 min with 5 µL annexinV/PI in a 100 µL binding buffer. Following dilution (500 µL binding buffer final volume) analysis was performed by flow cytometry using FacsCanto BD. The percentage of annexin V positive cells was evaluated by Flowjo BD Software, Milan, Italy.
Leica fluorescent microscope
Manufactured by Leica Microsystems
Sourced in Germany, Israel, United States
The Leica fluorescent microscope is a specialized lab equipment designed for the observation and analysis of fluorescently-labeled samples. It utilizes advanced optical technology to generate high-contrast, high-resolution images of fluorescent specimens, enabling researchers to study a wide range of biological and material science applications.
Automatically generated - may contain errors
Lab products found in correlation
Dapi containing mounting media, by Vector Laboratories (3 mentions)
Image pro software, by Media Cybernetics (3 mentions)
Vectashield medium, by Vector Laboratories (2 mentions)
Hoechst 33342, by Merck Group (2 mentions)
Fluorescein isothiocyanate (fitc), by Jackson ImmunoResearch (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Dojindo Laboratories (2 mentions)
Alexa fluor secondary antibody, by Thermo Fisher Scientific (2 mentions)
Leica q fluoro software, by Leica Microsystems (1 mentions)
Tetramethylrhodamine phalloidin, by Solarbio (1 mentions)
Cell counting kit 8 assay, by Dojindo Laboratories (1 mentions)
Microplate reader, by Bioteck (1 mentions)
Goat serum, by Nichirei Biosciences (1 mentions)
Anti fibronectin antibody, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Fujifilm (1 mentions)
Alexa fluor 488 conjugated goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Anti nestin antibody, by Merck Group (1 mentions)
Alexa fluor 546 conjugated goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Anti α sma antibody, by Merck Group (1 mentions)
Acetone, by Merck Group (1 mentions)
Hank s balanced salt solution, by Thermo Fisher Scientific (1 mentions)
20 protocols using leica fluorescent microscope
1
Apoptosis Evaluation by Hoechst and Annexin V
2
Osteocyte Viability and Morphology After Irradiation
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The cell viability of irradiated osteocytes was detected using a Cell Counting Kit-8 assay (CCK8; Dojindo Molecular Technologies, Inc.). Briefly, osteocytes seeded in 96-well plates (3×103 cells/well) were subsequently treated with irradiation (0, 2, 4 or 8 Gy), followed by incubation for 1, 3 or 5 days. CCK8 reagent was added at 10%, and incubated for 2 h at 37°C. The absorbance at 450 nm was examined using a microplate reader (Bioteck), and was considered to indicate cell viability.
The morphological changes including dendrite-like synapse and cytoskeleton in the irradiated osteocytes were examined by staining with tetramethyl rhodamine-phalloidin (Beijing Solarbio Science & Technology Co., Ltd.) for F-actin and with DAPI (Dojindo Molecular Technologies, Inc.) for nuclei. Each staining step was processed at room temperature and incubated for 2 h in the dark. The number and dendritic length of osteocytes were quantitatively measured using SimplePCI software (HCImage, SimplePCI 6.6). Six random fields were chosen in three biological replicates, and representative images were captured using a Leica fluorescent microscope (Leica Microsystems, GmbH) with a magnification of ×100. The length of the dendrites was calculated by total length of dendrites/number of dendrites per cell.
The morphological changes including dendrite-like synapse and cytoskeleton in the irradiated osteocytes were examined by staining with tetramethyl rhodamine-phalloidin (Beijing Solarbio Science & Technology Co., Ltd.) for F-actin and with DAPI (Dojindo Molecular Technologies, Inc.) for nuclei. Each staining step was processed at room temperature and incubated for 2 h in the dark. The number and dendritic length of osteocytes were quantitatively measured using SimplePCI software (HCImage, SimplePCI 6.6). Six random fields were chosen in three biological replicates, and representative images were captured using a Leica fluorescent microscope (Leica Microsystems, GmbH) with a magnification of ×100. The length of the dendrites was calculated by total length of dendrites/number of dendrites per cell.
3
Immunofluorescence Staining Protocol
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Cells were fixed with 4% paraformaldehyde (Wako) for 15 min, permeabilized with 0.1% Triton X-100 for 5 min and blocked with 10% goat serum (Nichirei Bioscience) in PBS for 1 hr at room temperature (RT). Cells were incubated with primary antibodies for 2 hr at RT before incubation with appropriate secondary antibodies for 1 hr at RT, prior to counterstaining with 4’,6-diamidino-2-phenylindole (DAPI)(Dojindo Laboratories). Samples were observed using a Leica fluorescent microscope (Leica Microsystems). The primary antibodies used were: anti-nestin antibody (1:100; Millipore), anti-fibronectin antibody (1:100; Sigma-Aldrich), anti-αSMA antibody (1:100; Sigma-Aldrich), anti-S100β antibody (1:100; Sigma-Aldrich) and anti-trichohyalin antibody (1:5; Santa Cruz Biotechnology). Secondary antibodies used were: Alexa Fluor 488-conjugated goat anti-mouse (1:200) and Alexa Fluor 546-conjugated goat anti-rabbit (1:200, both from Life Technologies).
4
Immunocytochemical Detection of Intracellular Tat
5
Histological Assessment of Severe Acute Pancreatitis
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Specimens of the pancreas was harvested and fixed in 10% formaldehyde solution for 48 h, dehydrated in graded alcohol series, cleared with xylene and embedded in paraffin. Tissue sections (4 µm) were stained with hematoxylin-eosin (H&E) for general morphology and observed under a Leica fluorescent microscope (Leica Microsystems, Wetzlar, Germany). The pathological grading described by Schmidt et al [23] (link) takes all factors, including pancreatic edema, acinar necrosis, hemorrhage and fat necrosis, as well as inflammation and perivascular infiltration, into consideration when scoring the pancreatic injury in experimental SAP [23] (link). The mean value of the total score of these four parameters in each group was analyzed. The pathological sections were examined by two experienced pathologists from the Department of Pathology, Wenzhou Medical College, in a blinded fashion.
6
Intracellular Calcium Measurement Protocol
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Intracellular calcium concentrations ([Ca 2þ ] i ) were measured with the calcium-sensitive dye Fluo-4/AM (Invitrogen Carlsbad, CA, USA) according to the manufacturer's protocol. Briefly, cells seeded on Ibidi (Martinsreid, Germany) 35-mm dishes were incubated with 2 mM Fluo-4/AM in Hanks' balanced salt solution (Invitrogen) at 22 °C for 45 min. Subsequently, cells were washed three times and further incubated with the latter solution at 22 °C for 30 min. Calcium measurements were performed with a Leica fluorescent microscope (Leica Microsystems, Wetzlar, Germany). Fluorescence was recorded at 1-s intervals for up to 20 min. At least 30 cells were counted under each condition. [Ca 2þ ] i was calculated using the following equation:
where F is the measured fluorescence intensity, Fmax is the fluorescence measured after addition of 10 mM ionomycin, Fmin is the fluorescence measured after addition of 10 mM EGTA, and K d is the dissociation constant of the dye-Ca 2þ complex at 22 °C (345 nM).
where F is the measured fluorescence intensity, Fmax is the fluorescence measured after addition of 10 mM ionomycin, Fmin is the fluorescence measured after addition of 10 mM EGTA, and K d is the dissociation constant of the dye-Ca 2þ complex at 22 °C (345 nM).
7
Apoptosis Quantification of Infected Cells
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Propidium iodide/acridine orange was used to investigate cell death by apoptosis of the infected and controlled AMGM5 cells. PI/AO dual-staining tests were performed. The cells (7 × 103 cells mL−1) were initially subjected to an overnight seeding in the 96-well plate and treated with IC50 concentrations of NDV (MOI 0.5), TMZ (3.125 μgmL−1), TMZ-PLGA-NPs (12.5 μgmL−1), NDV and TMZ (0.5 + 3.125), and NDV and TMZ-PLGA-NPs (0.5 + 12.5) with incubations at 37 °C for 72 h. The classical PI/AO staining protocol was then applied by adding 50 μL of AO/PI staining mixture to 1 mL of cell suspension. The preparations were then left to stand for 30 s at ambient temperature. After discarding the staining solution, the samples were directly photographed with the use of a Leica fluorescent microscope (Leica Microsystems, Wetzlar, Germany), and Image J software (http://rsb.info.nih.gov/ij/ accessed on 3 April 2022) was used to examine the images. Each photo was quantitatively measured in three different ways for statistical analysis. The percentage (%) of cells stained with green or red were determined [32 (link)].
8
Visualizing VEGFR2 Expression in HUVECs
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HUVEC cells were seeded on a slide in the 24-well plate. Immunofluorescence images of VEGFR2 expression on HUVECs were captured, after treatment with indicated media (Peptostreptococcus anaerobius treated macrophage media) stimulation for 72h. Cells were fixed with 10% paraformaldehyde, the VEGFR were stained by FITC-VEGFR antibody, and nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI) (Millipore) for 10 min at 4°C. Images were taken using a Leica fluorescent microscope and a TCS SP5 confocal laser scanning microscope (Leica Microsystems, Wetzlar, GER).
9
Fluorescence Microscopy of Nanocapsule Uptake
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The cells were seeded at concentration of 105 cells/mL in a 12-well plate containing glass coverslips (18 mm diameter) pretreated with 0.01% polylysine (Sigma-Aldrich, Saint Louis, MO, USA) and allowed to attach for 24 h. Then, cells were incubated with 10 µg/mL DiO encapsulated amphoteric nanocapsules (NC-DiO) at pH 7.8 for 1–48 h. The cells were then washed twice with PBS, incubated with 0.25 µM LysoTracker Red (DND-99) (Molecular Probes, Eugene, OR, USA) for 1 h, and fixed with 4% paraformaldehyde (PFA) for 20 min at 4 °C. Nuclei were dyed with Hoechst 33342 (Sigma-Aldrich, Saint Louis, MO, USA) and slides were mounted on Mowiol (Sigma-Aldrich, Saint Louis, MO, USA). The immunofluorescence images were captured using a Leica fluorescent microscope (Leica Microsystems, Wetzlar, Germany).
10
Quantifying Pancreatic β-cell Mass
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