To investigate Aβ plaque accumulation, immunofluorescence staining was conducted as previously described (28 (link)). Serial sections from the prefrontal cortex (PFC; bregma, 1.94–1.42 mm) to the hippocampus (bregma, − 1.70 to − 2.18 mm) were used for immunofluorescent staining. Three sections per animal were blocked with 5% normal goat serum (Vector Laboratories) and then immersed in anti-mouse 6E10 primary antibody (BioLegend) overnight at 4 °C. Sections were immersed in Alexa Fluor conjugated secondary antibody (Invitrogen) for 90 minutes, and incubated in 4′-6-diamidino-2-phenylindole solution (Sigma-Aldrich) for 10 minutes to stain the cellular nuclei. Stained sections were imaged using a fluorescence microscope (Olympus, Japan), and Aβ plaque load in the hippocampal subregions (CA1: cornu ammonis 1 and DG: dentate gyrus) and PFC (PL: prelimbic cortex and IL: infralimbic cortex) was quantified using Image J software (NIH). For Aβ plaque load analysis, the number of 6E10-positive pixels was divided by the number of whole pixels in each image and, according to previous reports (18 (link),29 (link)), the data are reported as the percentage area occupied by the 6E10-positive signal. To apply the same selection criteria to all cuts, a template was created from a region of interest in the hippocampus and PFC and applied on each sample.
4 6 diamidino 2 phenylindole solution
Manufactured by Merck Group
Sourced in Spain
4',6-diamidino-2-phenylindole (DAPI) solution is a fluorescent dye commonly used in research applications. DAPI selectively binds to the minor groove of DNA, allowing it to be used for staining and visualizing nucleic acids. The solution is provided in a liquid form for ease of use.
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Lab products found in correlation
Click it edu imaging kit, by Thermo Fisher Scientific (2 mentions)
Ckx41 f32fl, by Olympus (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Gallios flow cytometer, by Beckman Coulter (2 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Normal goat serum (ngs), by Vector Laboratories (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Bx41 fluorescence microscope, by Olympus (1 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (1 mentions)
Eclipse ti inverted light microscope, by Nikon (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Matrigel basement membrane matrix, by BD (1 mentions)
7 protocols using 4 6 diamidino 2 phenylindole solution
1
Quantifying Amyloid-Beta Plaque Burden
2
Alkaline Comet Assay for Genetic Damage
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The alkaline comet assay was performed as previously described by Collins and Azqueta [45 (link)]. Briefly, slides were precoated with low-melting-point agarose. For each animal, four slides were prepared (two for performing the assay with the repair enzyme and the other two for the assay without the enzyme). Approximately 10 μL of blood was diluted in 200 μL of ice-cold phosphate-buffered saline (PBS) in a 0.5 mL microtube to prepare a cell suspension. An aliquot of the cell suspension was placed onto the precoated slides. The slides were immersed in a lysis solution, and then electrophoresed. Following electrophoresis, the cells were immersed in PBS followed by distilled water, dehydrated in ethanol, and air-dried. The slides were then stained with 4,6-diamidino-2-phenylindole solution (Sigma-Aldrich Chemical Company, Spain) and observed using an Olympus BX41 fluorescence microscope (Tokyo, Japan) at 400×. The nucleoids were classified visually in five classes from 0 (no tail) to 4 (almost all DNA in the tail) (Collins, 2004). Fifty nucleoids were observed per mini-gel (100 per case). A genetic damage index (GDI), expressed in an arbitrary scale from 0 to 400, was obtained according to the formula:
3
Evaluating miR-3148 in Glioma Cell Proliferation
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After culturing cells at 37˚C for 24 h in a 96-well plate at a density of 2x103 cells/well, U87 MG and U251 cell lines were incubated at 37˚C for 2 h with 10 µl Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc.) solution. Then, the optical density at 450 nm was measured and recorded.
The role of miR-3148 in glioma cell proliferation was evaluated using Click-iT® EdU Imaging Kits (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. U87 MG and U251 cells were cultured in 96-well plates (8x103 cells/well) and incubated at room temperature with 10 µl of EdU reagent for 3 h. At room temperature, the cells were fixed with 4% formaldehyde for 20 min and washed with formaldehyde in PBS. The cells were then incubated at room temperature in 0.5% Triton X-100 (Sigma-Aldrich; Merck KGaA) for 5 min. The nuclei were then stained with a 4',6-diamidino-2-phenylindole solution (1 ml; Sigma-Aldrich; Merck KGaA), which was added to each well and incubated for 25 min at room temperature in the dark. The staining solution was then removed by washing thrice with PBS. Finally, the stained cells were imaged and counted under a fluorescence microscope (magnification, x100) (CKX41-F32FL; Olympus, Beijing, China). Each assay was performed in triplicates.
The role of miR-3148 in glioma cell proliferation was evaluated using Click-iT® EdU Imaging Kits (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. U87 MG and U251 cells were cultured in 96-well plates (8x103 cells/well) and incubated at room temperature with 10 µl of EdU reagent for 3 h. At room temperature, the cells were fixed with 4% formaldehyde for 20 min and washed with formaldehyde in PBS. The cells were then incubated at room temperature in 0.5% Triton X-100 (Sigma-Aldrich; Merck KGaA) for 5 min. The nuclei were then stained with a 4',6-diamidino-2-phenylindole solution (1 ml; Sigma-Aldrich; Merck KGaA), which was added to each well and incubated for 25 min at room temperature in the dark. The staining solution was then removed by washing thrice with PBS. Finally, the stained cells were imaged and counted under a fluorescence microscope (magnification, x100) (CKX41-F32FL; Olympus, Beijing, China). Each assay was performed in triplicates.
4
Evaluating miR-378a-3p's Role in HCC Proliferation
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As specified by the manufacturer’s protocol, Click-iT® EdU Imaging Kits (Invitrogen, Carlsbad, CA, USA) were employed to assess the role of miR-378a-3p in HCC cell proliferation. Cells (8 × 103 cells/well) were cultured in 96-well plates and incubated with 10 μL of EdU reagent for 3 h. Subsequently, cells were fixed with 4% formaldehyde for 20 min at room temperature, followed by rinsing with phosphate-buffered saline (PBS). The cells were then incubated in 0.5% Triton X-100 (Sigma, Shanghai, China) at room temperature. Nucleus staining was performed using 1 mL of 4’,6-diamidino-2-phenylindole solution (Sigma); the solution was added to each well and the cells were incubated for 25 min in the dark at room temperature. Then, the staining solution was removed by washing with PBS three times. Finally, pictures of the stained cells were taken, and the cells were quantified under a fluorescence microscope (CKX41-F32FL; Olympus, Beijing, China).
5
Ploidy Levels and Gonadal Histology
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Ploidy levels of the fish (RCC, 4nRR and 3nRR) were estimated using a flow cytometer (Gallios Flow Cytometer, Beckman Coulter). Blood was collected from the caudal vein using heparinized syringes. Samples were then resuspended in 4,6-Diamidino-2-Phenylindole solution (Sigma-Aldrich) for 10 min. DNA content was compared with that of RCC per sample. Gonadal tissues of two years old female RCC, male 4nRR and 3nRR were fixed in Bonn’s liquid and then dehydrated using graded series of alcohol, cleared with xylene, embedded in paraffin wax and cut into 5–8 μm sections. The sections were placed on slides, stained with hematoxylin and eosin, and viewed under a light microscope.
6
Quantifying Cellular Invasion Using Matrigel Transwell Assay
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A Matrigel-based Transwell system (8-mm pore size polycarbonate membrane; Corning, Inc.) was used to examine the invasiveness of transfected cells. The Transwell inserts were covered with a homogenous layer of BD Matrigel™ Basement Membrane Matrix (BD Biosciences) and incubated for 4 h at 37˚C in 24-well plates containing RPMI-1640 medium per 10-20 µl/well. Then, 0.3-1.0x105 cells/well in 250.0 µl serum-free medium were plated into the upper chamber and allowed to invade for 24-72 h at 37˚C. A total of 650.0 µl of RPMI 1640 medium containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) was plated in the lower chamber. The residual medium on the top insert was removed after the incubation time and non-invading cells on the upper surface were gently scraped with a brush. The migrating cells to the lower chambers were fixed in a fixing solution (4% formaldehyde in PBS with 0.1% Tween-20) at room temperature for 20 min after being washed twice with PBS. The cells were then stained for 10 min at 37˚C with 4',6-diamidino-2-phenylindole solution (Sigma-Aldrich; Merck KGaA) and photographed using a microscope Nikon ECLIPSE Ti inverted light microscope (Nikon Corporation) at 100x or 200x magnification. Subsequently, the number of invasive cells was by three fields per chamber using Image J software 1.52d (National Institutes of Health).
7
Ploidy Levels and Gonadal Histology Analysis
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Ploidy levels of the sh (RCC, 4nRR and 3nRR) were estimated using a ow cytometer (Gallios Flow Cytometer, Beckman Coulter). Blood was collected from the caudal vein using heparinized syringes. Samples were then resuspended in 4,6-Diamidino-2-Phenylindole solution (Sigma-Aldrich) for 10 min. DNA content was compared with that of RCC per sample. Gonadal tissues of two years old female RCC, male 4nRR and 3nRR were xed in Bonn's liquid and then dehydrated using graded series of alcohol, cleared with xylene, embedded in para n wax and cut into 5-8 μm sections. The sections were placed on slides, stained with hematoxylin and eosin, and viewed under a light microscope.
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