Doxorubicin cellular uptake was evaluated based on the cellular distribution of red auto-fluorescence of the drug. Briefly, drug-resistant U2OSR cells (1×105 cells per well) were cultured in 12-well plates for 24 h. Triplicate wells were treated with indicated doxorubicin and the indicated concentration of RA for 6 h, and images were then acquired with a fluorescence microscope (Leica). Drug efflux assays were performed as described 24 (link). Drug-sensitive and drug-resistant cells (1×105 cells per well) were cultured in 12-well plates for 24 h. Triplicate wells were treated with the indicated concentration of RA for 6 h and then exposed to 50 nM calcein AM. After a 30-min incubation, the cells were washed and fixed with 4% paraformaldehyde. The nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). Images were acquired with a fluorescence microscope (Leica). Cells were rinsed with DPBS, and total fluorescent emission in each well was measured with a SpectraMax® M5/M5e plate reader (Molecular Devices, US).
Fluorescence microscope
Manufactured by Leica camera
Sourced in Germany, United States, Japan, China, United Kingdom, India, Switzerland
The Leica Fluorescence Microscope is an advanced optical instrument designed for the observation and analysis of fluorescently labeled samples. It utilizes specific wavelengths of light to excite fluorescent dyes or proteins within the sample, causing them to emit light at a different wavelength, which is then captured and magnified by the microscope's optics.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Beyotime (62 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (56 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (48 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (34 mentions)
Triton x 100, by Merck Group (27 mentions)
In situ cell death detection kit, by Roche (25 mentions)
Bovine serum albumin (bsa), by Merck Group (15 mentions)
Hoechst 33342, by Merck Group (13 mentions)
4 6 diamidino 2 phenylindole (dapi), by Solarbio (13 mentions)
Vectashield, by Vector Laboratories (12 mentions)
Triton x 100, by Beyotime (12 mentions)
Paraformaldehyde (pfa), by Merck Group (11 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (11 mentions)
One step tunel apoptosis assay kit, by Beyotime (10 mentions)
Hoechst 33342, by Thermo Fisher Scientific (10 mentions)
Hoechst 33342, by Beyotime (9 mentions)
Fish kit, by RiboBio (9 mentions)
Ab150077, by Abcam (8 mentions)
Ros assay kit, by Beyotime (8 mentions)
4 6 diamidino 2 phenylindole (dapi), by Wuhan Servicebio Technology (8 mentions)
1 806 protocols using fluorescence microscope
1
Doxorubicin Uptake and Efflux in Drug-Resistant Cells
2
Apoptosis and Neuronal Colocalization Analysis
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Double label staining of TUNEL with neuron-specific nuclear protein (NeuN) was performed to explore colocalization of apoptosis cells and neurons. Briefly, the rats were perfused with 4% paraformaldehyde and postfixed by immersion in the same fixative overnight. Then the brains were paraffin-embedded, and sectioned at a thickness of 10 µm. TUNEL staining was performed according to the manufacturer’s protocol of the commercial kit (Roche, Switzerland), followed by antibody staining against NeuN (1∶200, Abcam, UK). Finally, the sections were covered with 4′6-diamidino-2-phenylindole (DAPI) (Beyotime, China) and observed under a fluorescence microscope (Leica, Germany).
Double staining of pAkt (Ser-473) with TUNEL staining was performed to clarify the relationship between apoptosis and pAkt (Ser-473). The sections were stained with the antibody against pAkt (serine-473) (1∶200, Cell signaling Technology, USA), followed by TUNEL staining. Then the sections were covered with DAPI (Beyotime, China) and observed with a fluorescence microscope (Leica, Germany).
Double staining of pAkt (Ser-473) with TUNEL staining was performed to clarify the relationship between apoptosis and pAkt (Ser-473). The sections were stained with the antibody against pAkt (serine-473) (1∶200, Cell signaling Technology, USA), followed by TUNEL staining. Then the sections were covered with DAPI (Beyotime, China) and observed with a fluorescence microscope (Leica, Germany).
3
Fluorescent Glucose and Lipid Quantification
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Glucose uptake was analyzed directly using the fluorescent glucose analog 2-NBDG. HCC cells were incubated in glucose-free RPMI medium containing 100 mM 2-NBDG for 90 min at 37 °C in dark, and the amount of 2-NBDG taken up by cells was assessed by flow cytometry analysis (FACS). For fluorescence examine, HCC cells grown on coverslips were fixed with 4% paraformaldehyde for 10 min, then cells were washed 3 times in cold PBS and stained with 100 mM 2-NBDG for 90 min at 37 °C in dark, and cell images were photographed using a fluorescence microscope (Leica, Wetzlar, Germany). For lipid content measurement , 2×105 cultured HCC cells were incubated in the presence of 1 μM fluorescent lipid probe Bodipy 558/568 for 30 min at 37 °C in dark. Then the labeled cells were washed and re-suspended in cold PBS and the lipid content was quantified using FACS as mentioned above. For fluorescence examine, HCC cell lines grown on coverslips were fixed with 4% paraformaldehyde for 10 min. Following fixation, cells were washed 3 times in cold PBS and stained with 1 μM Bodipy 558/568 for 30 min at 37 °C in dark, and cell images were photographed using a fluorescence microscope (Leica).
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4
Exosome Tracking in Lung Inflammation
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For confocal experiments, ADSCs were transfected with cyc3-labelled oligo to obtain exosomes. Then, the BMDM were treated with exosomes containing cyc3-labelled oligo and GW4869 (exosomal inhibitor). The cells were observed and photographed under a fluorescence microscope (Leica, Germany). To determine the localization of exosomes in the lung, the lung sections were incubated with anti-CD86 antibody (biomarker of macrophages) and anti-Ly-6G antibody (biomarker of neutrophiles), followed by incubation with Alexa 488 secondary antibody (Thermo) and DAPI. The images were photographed by the fluorescence microscope (Leica). Mouse lung tissues were also stained by haematoxylin and eosin (H&E) dye to observe tissue damage and captured by a microscope (Leica).
5
Apoptosis Assays: Hoechst and Annexin V
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Apoptosis was monitored with the Hoechst assay and with the Annexin V binding assay. For both assays, cells were plated in 96-well plates (5,000 cells/well) and, after 24 hrs, treated for 48 hrs as described in the figure legends.
For the Hoechst assay, the cells were stained with 5 μg/ml Hoechst 33342 (10 min, 37 °C), and both floating and adherent cells were analyzed by using a hemocytometer under a fluorescence microscope (Leica). Cells incorporating the Hoechst dye and showing typical morphological apoptotic features, such as chromatin condensation, were considered apoptotic cells, according to Schmid et al.26 (link). Data were expressed as the apoptosis index ((apoptotic cells/total cells) × 100).
For the Annexin V assay, treated cells were stained using the annexin V–FITC fluorescence microscopy kit (BD Biosciences), according to the manufacturer’s instructions. Apoptotic cells were finally detected with a fluorescence microscope (Leica) equipped with an online image capture system (LeicaDFC320).
For the Hoechst assay, the cells were stained with 5 μg/ml Hoechst 33342 (10 min, 37 °C), and both floating and adherent cells were analyzed by using a hemocytometer under a fluorescence microscope (Leica). Cells incorporating the Hoechst dye and showing typical morphological apoptotic features, such as chromatin condensation, were considered apoptotic cells, according to Schmid et al.26 (link). Data were expressed as the apoptosis index ((apoptotic cells/total cells) × 100).
For the Annexin V assay, treated cells were stained using the annexin V–FITC fluorescence microscopy kit (BD Biosciences), according to the manufacturer’s instructions. Apoptotic cells were finally detected with a fluorescence microscope (Leica) equipped with an online image capture system (LeicaDFC320).
6
Visualizing PAM's Cytotoxic Effects on Lung Cancer Cells
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To visually observe the killing effect of PAM, the lung cancer cells were seeded onto a 24-well plate with a cell density of 5 × 104 cells/well and incubated overnight. The cells were then treated with or without PAM for 48 h. Then, the cells were washed with phosphate-buffered saline (PBS), followed by staining with calcein-AM/PI double-staining kit (Yeasen, Shanghai, China) following the manufacturer's instructions. Briefly, 2 μM of calcein-AM and 4.5 μM of PI were added to the cells and incubated for 15 min at 37°C, followed by observation under a fluorescence microscope (Leica, Germany). The relative intensity of PI-indicating dead cells was analyzed using ImageJ software of images in five independent experiments.
For 3D spheroids, after spheroid formation (at day 4), the culture medium was changed to PAM (20 min activated) and treated for 48 h. Then, a similar staining process was performed using calcein-AM and PI. The images were taken using a fluorescence microscope (Leica, Germany). The relative intensity of PI-indicating dead cells was analyzed using ImageJ software.
For 3D spheroids, after spheroid formation (at day 4), the culture medium was changed to PAM (20 min activated) and treated for 48 h. Then, a similar staining process was performed using calcein-AM and PI. The images were taken using a fluorescence microscope (Leica, Germany). The relative intensity of PI-indicating dead cells was analyzed using ImageJ software.
7
Evaluating BMSC Viability and Morphology on Titanium Surfaces
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Sterilized Ti plates were placed in a 24‐well plate. Each well was inoculated with 2 × 104 BMSCs and cells inoculated on tissue culture plastic were used as NC. Cell viability on different substrates was detected by live/dead staining. After 24 h of culture, the cells were stained with Calcein‐AM (2 µm ) and PI (4.5 µm ), and observed under a fluorescence microscope (Leica). Cell morphology on different samples was observed by SEM (Pro G5, Phenome) after fixation with Gluta fixative (Solarbio), gradual dehydration, lyophilization, and gold sputtering. Furthermore, FITC‐phalloidin was used to mark F‐actin to observe the cytoskeleton. Briefly, after 24 h of incubation, cells were fixed with 4% PFA for 10 min and permeabilized with 0.1% Triton X‐100 in PBS 3 times for 5 min each. Subsequently, cells were stained with FITC‐phalloidin (1:100 diluted) plus DAPI (5 µg mL−1) and subjected to a fluorescence microscope (Leica). Additionally, cell proliferation was evaluated on 1, 2, and 3 days with the Cell Counting Kit‐8 (CCK‐8, Dojindo, Tokyo, Japan) according to the procedures provided by the manufacturer.
8
Immunofluorescent Staining of Cells and Tissue
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OM-MSCs laid on orifice plates were fixed with 4% paraformaldehyde and incubated with primary antibody. The following antibodies are used for staining: NeuN (1/300; ab177487, Abcam, Cambridge, MA, USA) and GFAP (1/5000; ab7260, Abcam). Then, cells were incubated with corresponding secondary antibodies, and nuclei were stained with DAPI (Beyotime, Shanghai, China). A fluorescence microscope (Leica, Wetzlar, Germany) was used to acquire images for analysis. Frozen sections (4 μm thick) were obtained from the cerebral hemorrhage area of each animal, infiltrated with 0.5% Triton X-100, and incubated overnight with NueN (1:100) antibody at 4°C. Alexa Fluor488-conjugated goat rabbit IgG was used to localize signals. Finally, DAPI was used to reverse stain the sections and images were observed under a fluorescence microscope (Leica).
9
Immunofluorescence Staining of T-Cell Subsets
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Paraffin sections were de-paraffinized, rehydrated and antigen retrieval according to standard protocols. Then, slices were incubated with anti-CD3 (1:200, Abcam, ab16669), anti-CD4 (1:200, Abcam, ab237722), anti-CD8 (1:200, Abcam, ab33786), and anti-Foxp3 (1:100, Abcam, ab215206). Fluorophore-conjugated secondary antibodies were incubated for one hour at room temperature (1:200, Abcam). The slides were imaged with fluorescence microscopes (Leica, Barcelona, Spain).
10
Immunofluorescence Analysis of Hippocampal Cells
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After adjusting the hippocampal cells to 1.5 × 104/100 µL, they were centrifuged for 10 minutes to make them adhere to the slides, and later fixed with 4% paraformaldehyde for 15 minutes. The slides were incubated with 0.25% bovine serum albumin for 5 minutes, and incubate with 0.05% Triton X-100 on the slides for 5 minutes. After incubating with anti-VEGF (1: 1,000, ab46154, Abcam, U.S.), anti-βIII tubulin (1: 1000, G7121, Promega) at 4°C overnight, the slides mixed with green fluorescence-labeled goat anti-rabbit secondary antibody (1: 1000, sc-2012, Santa Cruz, U.S.) were placed in the darkness at 37°C for 60 minutes, where the nucleus was stained with 4ʹ,6-Diamidino-2-phenylindole (DAPI) for 10 minutes, later the slides observed under fluorescence microscopes (Leica, Germany).
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