Cells were seeded into 96-well plates and 100 μL of EdU reagent was added and cultured for 2 h. After washing with PBS (Solarbio), the cells were dyed with Apollo and then washed with methanol (Solarbio). Later, the cells were fixed, discolored and permeated. Subsequently, the cells were cultivated with 100 μL of DAPI reaction solution (Sigma-Aldrich Chemical Company, St Louis, MO, USA) followed by viability determination via a fluorescent microscope (Thermo Fisher Scientific).
Fluorescent microscope
Manufactured by Thermo Fisher Scientific
Sourced in United States, Japan
A fluorescent microscope is an optical instrument used to visualize and study fluorescent samples. It utilizes specific wavelengths of light to excite fluorescent molecules within the sample, causing them to emit light at different wavelengths that can be detected and imaged.
Automatically generated - may contain errors
Lab products found in correlation
Dapi reaction solution, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Solarbio (1 mentions)
Methanol, by Solarbio (1 mentions)
Crystal violet, by Merck Group (1 mentions)
Matrigel, by BD (1 mentions)
Rabbit anti nrf2 antibody, by Santa Cruz Biotechnology (1 mentions)
Ab34710, by Abcam (1 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
A 11072, by Thermo Fisher Scientific (1 mentions)
A27034, by Thermo Fisher Scientific (1 mentions)
Oct compound, by Sakura Finetek (1 mentions)
Ab5694, by Abcam (1 mentions)
Triton x 100, by Solarbio (1 mentions)
Bovine serum albumin (bsa), by Avantor (1 mentions)
Ribo fish kit, by RiboBio (1 mentions)
Crnde, by RiboBio (1 mentions)
Paris kit, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 labeled anti rabbit secondary antibody, by Thermo Fisher Scientific (1 mentions)
Mounting medium, by Thermo Fisher Scientific (1 mentions)
Dii ox ldl, by Biomedical Technologies (1 mentions)
12 protocols using fluorescent microscope
1
Cell Proliferation Assay with EdU
2
Quantifying Microbial Biofilm Viability
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For qualitative assessment of microbial biofilms, viable biofilm cells staining by acridine orange was used. Eighty samples of CORM were collected from the twenty-saliva collectors. The samples of CORM were stained with 1 ml acridine orange acidic stain stock solution (ThermoFisher Scientific, Waltham, Germany). Acridine orange acidic stain was prepared by dissolving 50 mg of acridine orange in 10 ml of distilled water to prepare a reserve solution. To prepare a working solution, 1 ml of Acridine orange stock solution mixed with 0.5 ml of glacial acetic acid and 50 ml distilled water. The biofilm on the samples was fixed with methanol, dried, and stained with acridine orange staining working solution for 2 min. The samples were washed gently with water, dried, and then examined using a fluorescent microscope (Thermo Fisher Scientific, Waltham, Germany) under a high-power magnification oil lens (100*10x).
3
Transwell Invasion Assay of A549 Cells
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A total of 1×105 A549 cells were plated in the upper chambers of Transwell plates in serum-free medium for 24 h. Transwell membranes were precoated with Matrigel (Becton) at 37°C for 2 h. Medium supplemented with 20% FBS was plated in the lower chambers. Cells in the upper chambers were discarded using cotton swabs, while the migratory cells in the lower chambers were fixed with 4% methanol, prior to staining with 0.1% crystal violet (Sigma-Aldrich; Merck KGaA). Stained cells were counted in randomly selected fields using a fluorescent microscope (Thermo Fisher Scientific, Inc.; magnification, ×100).
4
Immunofluorescence Analysis of Nrf2 Localization
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Cells were grown on
fibronectin-coated coverslips in the presence or absence of 6S. After
treatment with test samples for the indicated times, cells were fixed
with cold 2% (w/v) paraformaldehyde for 20 min, permeabilized in 0.1%
(w/v) Triton X-100 in 1× PBS, washed, and blocked in 1% BSA at
room temperature for 1 h. Tissue sample sections were deparaffinized
and rehydrated. After being washed with PBS for 5 min three times,
the cells were incubated with a rabbit anti-Nrf2 antibody (1:200;
Santa Cruz Biotechnology) and tissue sections were incubated with
a rabbit anti-Nrf2 antibody (1:200)39 (link) overnight
at 4 °C, followed by FITC-conjugated secondary antibody (1:200)
for 1 h at room temperature. Samples were counterstained with DAPI
(1 mg/mL) for visualization of the nuclei. Stained samples were mounted
and visualized under a fluorescent microscope (Thermo Fisher Scientific).
fibronectin-coated coverslips in the presence or absence of 6S. After
treatment with test samples for the indicated times, cells were fixed
with cold 2% (w/v) paraformaldehyde for 20 min, permeabilized in 0.1%
(w/v) Triton X-100 in 1× PBS, washed, and blocked in 1% BSA at
room temperature for 1 h. Tissue sample sections were deparaffinized
and rehydrated. After being washed with PBS for 5 min three times,
the cells were incubated with a rabbit anti-Nrf2 antibody (1:200;
Santa Cruz Biotechnology) and tissue sections were incubated with
a rabbit anti-Nrf2 antibody (1:200)39 (link) overnight
at 4 °C, followed by FITC-conjugated secondary antibody (1:200)
for 1 h at room temperature. Samples were counterstained with DAPI
(1 mg/mL) for visualization of the nuclei. Stained samples were mounted
and visualized under a fluorescent microscope (Thermo Fisher Scientific).
5
Evaluating Mitochondrial Membrane Potential
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The MMP was evaluated through JC‐1 staining, utilizing a previously established protocol.[34 (link)
] Neurons were stimulated with OxyHb and treated with or without celastrol for 24 h. After being washed 3 times with PBS, the cells were incubated in a prepared JC‐1 working solution at 37 °C for 30 min. Subsequently, the stained cells were visualized under a fluorescent microscope (Tokyo, Japan), and fluorescence intensity was quantified using a fluorescent microplate reader (excitation/emission wavelengths of 514/529 nm for monomers and 585/590 nm for aggregates) (SpectraMax Paradigm, Thermo Fisher Scientific, USA).
] Neurons were stimulated with OxyHb and treated with or without celastrol for 24 h. After being washed 3 times with PBS, the cells were incubated in a prepared JC‐1 working solution at 37 °C for 30 min. Subsequently, the stained cells were visualized under a fluorescent microscope (Tokyo, Japan), and fluorescence intensity was quantified using a fluorescent microplate reader (excitation/emission wavelengths of 514/529 nm for monomers and 585/590 nm for aggregates) (SpectraMax Paradigm, Thermo Fisher Scientific, USA).
6
Immunofluorescence Imaging of Lung Tissue
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The lung tissue was harvested, trimmed and embedded in OCT compound (Sakura Finetek USA, Inc., Torrance, CA, USA) at −20°C for 20 min. The lung tissue was then cut into slices at a thickness of 5 µm. The slices were incubated with antibodies against α-SMA (ab5694; Abcam, Cambridge, MA, USA) and collagen I (ab34710; Abcam). Following incubation with secondary antibodies conjugated with Alexa 594 (A-11072; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and Alexa 488 (A27034; Invitrogen; Thermo Fisher Scientific, Inc.), respectively, fluorescent images were observed under a fluorescent microscope (Nikon, Tokyo, Japan).
7
Immunofluorescence Assay for DNA Damage
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A total of 2 × 104 cells was grown with cell culture medium and treated with designated concentrations of HJC0152 on chambered slides. At designated times after treatment, cells were fixed with 4% paraformaldehyde in PBS and then permeabilized with 0.1% Triton X‐100 (Solarbio) in PBS for 20 minutes at room temperature. Cells were subsequently washed and blocked with 5% BSA (Amresco) in PBS for 60 minutes at room temperature. Primary antibodies against γ‐H2AX were applied to the cells and incubated at 4°C overnight. After being washed with PBS 3 times, the cells were incubated with fluorescence‐conjugated secondary antibodies for 60 minutes at room temperature and then washed 3 times with PBS. Cell nuclei were counterstained with DAPI. Cells were visualized using a fluorescent microscope (Thermo Fisher) or confocal microscope.
8
Localization and Expression of CRNDE in Glioma
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For FISH assay, first, the fluorescent probe was synthesized on CRNDE, which was purchased from RiboBio (Guangzhou, China). Afterward, Ribo FISH Kit and Ribo lncRNA FISH Probe Mix Kit were used to perform FISH assay (RiboBio, Guangzhou, China) on brain glioma tissues in accordance with the instructions. After the assay, the fluorescent microscope was used to observe the results (Thermo Fisher, USA) and photographed. The cell nucleus and cytoplasm of T98G and U251 were extracted with PARIS Kit according to the kit specifications (Life Technologies, Carlsbad, USA). After the separation, the expressions of CRNDE and reference genes (U6 and GAPDH) in the cell nucleus and cytoplasm were assayed via qRT-PCR.
9
Immunofluorescence Detection of Active Caspase-3
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For detecting c-Caspase-3 using immunofluorescence, MC3T3-E1 cells were initially plated onto chamber slides. After removal of the blocking solution, the cells were incubated with the specific antibody, as indicated, at 4 °C overnight. Anti-c-Caspase-3 antibody were used at 1:300 dilution. For immunofluorescence detection, the cells were incubated with Alexa Fluor 488-labeled anti-rabbit secondary antibody (Invitrogen). For nuclear staining, the cells were incubated with 1 mg/mL 4′,6′-diamino-2-phenylindole (DAPI) for 5 min. The cells were fixed 2.5% formalin and stained with Hoechst staining. Fluorescence images were collected by using fluorescent microscope (ThermoFisher Scientific).
10
Visualizing Lysosomal Uptake of Oxidized LDL
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Human fetal RPE and ARPE-19 cells were grown for at least 1 week on laminin-coated 12-mm coverslips and were incubated with 10 μg/mL DiI-ox-LDL (Biomedical Technologies, Alfa Aesar, LLC, MA, USA) at 37°C with 5% CO2 for 15 hours. The cells were washed and fixed with 4% paraformaldehyde, followed by blocking (as above) for 1 hour at RT. The cells were then incubated overnight at 4°C with anti–lysosomal membrane associated protein (LAMP)-1 antibody, washed and treated with respective secondary antibody for 2 hours at RT, rinsed in PBS, mounted using mounting medium (Life Technologies); and imaged using a fluorescent microscope (Nikon Corp.).
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