After the OGD experiments, microglial cells were subjected to OGD treatment and treated with 4.5% glucose containing original medium as controls. Then, cellular ROS levels were detected using CellRox Green Reagent (C10444: Thermo Fisher Scientific). Briefly, the microglial cells were stained with 5 μM CellRox Green Reagent by adding the probe to the complete media and incubating at 37 °C for 30 min. Fifty micromolar of N-acetyl cysteine (NAC), an antioxidant was added to some of the OGD-treated wells. Then, the cells were washed with PBS and fixed with 4% PFA. Finally, the microglial nuclei were stained with Hoechst 33342 (Roche). The cells were observed with a microscope (IX71: Olympus, Tokyo, Japan), and the mean fluorescent intensities per cell were analyzed using an In Cell Analyzer 2000 (GE Healthcare).
Hoechst 33342
Manufactured by Roche
Sourced in Germany, Switzerland, United States
Hoechst 33342 is a fluorescent dye commonly used in cell biology applications. It binds to the minor groove of DNA, allowing visualization of nuclei in living or fixed cells.
Automatically generated - may contain errors
Lab products found in correlation
In cell analyzer 2000, by GE Healthcare (2 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
Tcs sp5, by Leica (2 mentions)
Cellrox green reagent, by Thermo Fisher Scientific (1 mentions)
Proteinase k, by Roche (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
In situ cell death detection pod kit, by Roche (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Dulbecco phosphate buffered saline (dpbs), by Corning (1 mentions)
Spe confocal microscope, by Leica (1 mentions)
Cytofix, by BD (1 mentions)
Fibronectin, by Abcam (1 mentions)
Vimentin, by Novus Biologicals (1 mentions)
Alexa fluor 488 conjugated goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Er tracker red, by Thermo Fisher Scientific (1 mentions)
Fluorescent microscope, by Olympus (1 mentions)
Sp5 confocal microscope, by Leica (1 mentions)
Iba 1, by Proteintech (1 mentions)
Alexa fluor 488 labeled anti mouse igg, by Thermo Fisher Scientific (1 mentions)
12 protocols using hoechst 33342
1
Microglial Oxidative Stress Measurement
2
Paraffin-Embedded Tissue TUNEL Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The tumors reserved in 4% formalin were embedded in paraffin and sectioned into thick slices, then incubated with proteinase K, TUNEL, and Hoechst 33342 following the manufacturer's instructions (Roche, Mannheim, Germany).
3
Immunofluorescence Microscopy Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were fixed in 4% paraformaldehyde (Sigma-Aldrich, Germany) for 10 min, and permeabilized in PBS containing 0.1% Triton X-100 (Sigma-Aldrich) for 10 min. After treatment with 5% BSA blocking for 1 h at room temperature, the primary antibody incubations were performed overnight at 4°C followed by the appropriate Alexa Fluor-conjugated secondary antibodies (Invitrogen, USA) for 2 h at room temperature, and nuclei were stained with Hoechst 33342 (Roche, Switzerland). Images were acquired using a confocal microscope (TCS-SP5, Leica).
4
Apoptosis Analysis in RF-EMF-Exposed eNSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell apoptosis was analysed by TUNEL and Hoechst 33342 staining. The cultured eNSCs were exposed to a 4 W/kg RF-EMF for 3 days. The cells were then dissociated and plated onto poly-L-lysine-coated round coverslips (12 mm in diameter) at a concentration of 1.0 × 105 cells/ml. Six hours after attachment, the eNSCs were harvested and fixed with 4% paraformaldehyde for 20 min. Then, the cells were rinsed in PBS and were stained with TUNEL or Hoechst 33342.
The TUNEL assay was performed using an in situ cell death detection POD kit (Roche Diagnostics Corp., USA) following the manufacturer's instructions. TUNEL-positive cells were counted in four non-overlapping fields per coverslip using a 40× objective. For each condition, more than 1000 cells in 12 coverslips from four independent experiments were counted. Data were converted to percentages of the total cell numbers.
For nuclear morphology staining, Hoechst 33342 (Sigma-Aldrich, USA) was applied to the cells at a concentration of 5 μg/ml for 30 min at 37°C. Then, the cells were washed with PBS and examined under a Leica fluorescence microscopy (Leica CTR6000, Germany). Apoptotic nuclei were identified by morphological changes, such as chromatin condensation and nuclear fragmentation. Data were obtained as described in the TUNEL assay section.
The TUNEL assay was performed using an in situ cell death detection POD kit (Roche Diagnostics Corp., USA) following the manufacturer's instructions. TUNEL-positive cells were counted in four non-overlapping fields per coverslip using a 40× objective. For each condition, more than 1000 cells in 12 coverslips from four independent experiments were counted. Data were converted to percentages of the total cell numbers.
For nuclear morphology staining, Hoechst 33342 (Sigma-Aldrich, USA) was applied to the cells at a concentration of 5 μg/ml for 30 min at 37°C. Then, the cells were washed with PBS and examined under a Leica fluorescence microscopy (Leica CTR6000, Germany). Apoptotic nuclei were identified by morphological changes, such as chromatin condensation and nuclear fragmentation. Data were obtained as described in the TUNEL assay section.
5
Multiparameter Immunofluorescence Staining
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were fixed with BD Cytofix (BD Bioscience) for 10 min at 22 °C, washed twice with dPBS (Cellgro) and permeabilized in dPBS + 0.2% Triton X-100 for 1 h at 22 °C followed by two additional dPBS washes. For antigen recovery, required for SP5 staining, cells were incubated with dPBS + 1% Sodium Dodecyl Sulfate for 10 min at 22 °C and washed three times with dPBS. Cells were blocked by treatment with buffer (dPBS, 0.5 mM EDTA, 1% BSA) for 30 min at 22 °C. Primary antibodies (see below) were diluted in buffer and incubated with cells overnight at 4 °C, followed by two washes with FACS buffer. Secondary antibodies were diluted in buffer and incubated with cells for 1 h at 22 °C, followed by two washes with FACS buffer. Nuclei were labeled with Hoechst 33342 (Roche) diluted 1:10,000 in buffer, followed by two washes with buffer. Cells were imaged on a Leica SPE confocal microscope.
6
Characterization of Cell Morphology and Surface Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cell morphology and surface protein expression of the two types of cells were compared. Morphologic observations of the third passage dADSCs and VFFs were made using an inverted microscope. The cell surface protein expression levels were identified by cell immunofluorescence.
When the cells reached 80% to 90% confluence, they were fixed in ice-cold 4% paraformaldehyde for 10 minutes and rinsed with phosphate-buffered saline (PBS). They were permeabilized in PBS containing 0.3% Triton X-100 for 30 minutes at room temperature (RT). All cells were blocked (3% horse serum in PBS, 30 minutes, RT) and incubated for 2 hours (4°C) with the following primary antibodies: vimentin (monoclonal mouse vimentin, 1∶200; Novus, Littleton, CO, USA) and fibronectin (polyclonal rabbit fibronectin, 1∶100; Abcam, Cambridge, MA, USA). After a thorough washing, the cells were incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG (1∶400; Molecular Probes, Eugene, OR, USA) and 594-conjugated goat anti-mouse IgG (1∶400; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 1 hour (RT). All slides were counterstained using Hoechst 33342 (1∶1000; Roche; 10 minutes, RT) for nuclear staining.
When the cells reached 80% to 90% confluence, they were fixed in ice-cold 4% paraformaldehyde for 10 minutes and rinsed with phosphate-buffered saline (PBS). They were permeabilized in PBS containing 0.3% Triton X-100 for 30 minutes at room temperature (RT). All cells were blocked (3% horse serum in PBS, 30 minutes, RT) and incubated for 2 hours (4°C) with the following primary antibodies: vimentin (monoclonal mouse vimentin, 1∶200; Novus, Littleton, CO, USA) and fibronectin (polyclonal rabbit fibronectin, 1∶100; Abcam, Cambridge, MA, USA). After a thorough washing, the cells were incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG (1∶400; Molecular Probes, Eugene, OR, USA) and 594-conjugated goat anti-mouse IgG (1∶400; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 1 hour (RT). All slides were counterstained using Hoechst 33342 (1∶1000; Roche; 10 minutes, RT) for nuclear staining.
7
Visualizing hBMSCs with GFP nanoparticles
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The hBMSCs were cultured in 35-mm cell dishes (Falcon, USA) at the density of 60%, transduced by GFP nanoparticles with a molar ratio of 16:1 to 2:1 (PEI1200/GFP) for 24 h. Nuclei were stained with Hoechst 33342 (Roche, Switzerland). Fluorescent images of living cells were acquired using a confocal microscope (TCS-SP5, Leica). For alternative protocol, cells were incubated with ER Tracker Red (Thermo Fisher, USA) for 30 min following the manufacturer’s instructions.
8
Assessing Apoptosis in PC12 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Chromosomal condensation and morphological changes in the nucleus were observed by using the chromatin dye, Hoechst 33342 (Roche®). Cells with homogeneously stained nuclei were considered to be viable. Chromatin condensation or fragmentation indicated apoptosis. Briefly, PC12 cells (1×106) were plated in 6 well plates and cultured on cover slips (pretreated with poly-D-lysine) with three mL of medium in each well. After 24 hours the cells were exposed to 500 μM of H2O2 with various concentrations of L-arginine for additional 24 h. After treatments, the cells were fixed with 4% formaldehyde in PBS for 20 min at room temperature and stained with one μg/mL Hoechst 33342 for 20 min. Cells were photographed under a fluorescent microscope (Olympus, Tokyo, Japan) and quantified by ImageJ 1.51 software. In each image, identical rectangular regions of interest with the selection tool of ImageJ were selected randomly, and the average pixel intensity was measured by the software.
9
Immunofluorescence Characterization of Airway Epithelial Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cultured NHBEs on transwells and matrigel were briefly washed three times with PBS and fixed in 4% paraformaldehyde for 10 min. The cells were then permeabilized with 0.2% Triton X‐100 in PBS for 20 min and blocked in goat serum for 1 h at room temperature (RT). The cells were incubated with TP63 (Cat. No. V3815SAF, NSJ Bioreagents) and FOXJ1 (cat. No. 14‐9965‐80. Thermo Scientific, MA, USA) antibodies overnight at 4°C and subsequently incubated with FITC‐conjugated goat anti‐rabbit and Texas red conjugated goat anti‐mouse antibodies for 1 h. The cells were washed with PBS, and the nuclei were stained with Hoechst 33342 (Roche Molecular Biochemicals) for 5 min at RT. Immunofluorescence signals were analyzed determined by confocal laser scanning microscopy (Leica SP5 Confocal Microscope, Leica Microsystems Inc.).
10
Immunostaining of Brain Cryosections
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Rats were re-anesthetized and transcardially perfused with normal saline, followed by 4% paraformaldehyde (PFA) in 0.1 M PBS. The brain was removed and immersed in the same fixative at 4 °C for 24 h and then transferred into 15% sucrose in PBS for 24 h and 30% sucrose in PBS for 24 h and embedded in O.C.T. compound. The embedded tissue was immediately frozen in liquid nitrogen and stored at -20 °C until use. The coronal sections of the brain frozen sections were cut with a cryostat at 15 µm, and mounted onto glass slides, as previously described. For immunofluorescence staining, brain sections were permeabilized with 0.01% Triton X-100 and 10% normal goat serum in PBS, pH 7.4, for 60 min. Then the sections were stained with primary antibodies against Iba-1 (1:200; microglia marker; rabbit, Proteintech), GFAP (1:500; astrocyte marker, rat, Dako). The sections were then incubated with Alexa Fluor-conjugated secondary antibodies (1:250; Invitrogen). Nuclear counterstaining was performed using Hoechst 33342 (1:1000; Roche).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!