PC12 cells were plated into 24-well plates at a density of 2,000 cells/well. After 7 days of differentiation as described above, the immunofluorescence was assessed to evaluate the neurite density. First, cells were fixed with 4% paraformaldehyde at room temperature for 60 min and washed with 0.2% Triton X-100 three times. Subsequently, PC12 cells were immunostained at 4°C overnight with the anti-MAP2 antibody (dilution 1:1,000) containing 1% goat serum and 0.5% Triton X-100, and the cells were washed again with 0.2% Triton X-100, followed by incubation with goat anti-rabbit IgG H&L (dilution 1:500, secondary antibody). Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, 1 μg/mL). The fluorescent images were visualized using a fluorescence microscope system (BX61/DP70, Olympus). ImageJ software (NIH, Bethesda, MD, United States) was used for quantitative analysis.
Bx61 dp70
Manufactured by Olympus
Sourced in Japan
The BX61/DP70 is a microscope system composed of the BX61 motorized microscope body and the DP70 digital camera. The BX61 provides high-quality optics and motorized control for various functions, while the DP70 captures digital images of the samples being observed through the microscope.
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8 protocols using bx61 dp70
1
Neurite Density Quantification in PC12 Cells
2
Evaluating GSK-3β Activation in Aβ-Treated Cortical Neurons
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Mouse cortical neurons (E14) were cultured in 8-well chamber slides at a density of 1.45 × 104 cells/cm2 for 3 days. KKT (10 μg/mL) or vehicle was administered to the cells, and Aβ (25–35) (10 μM) was added 1 h later. At 4 h after incubation with Aβ, the cells were fixed with 4% paraformaldehyde and immunostained with a monoclonal antibody against phosphorylated GSK-3β at Tyr-216 (pY216, dilution 1 : 200, BD Biosciences, Franklin Lakes, NJ, USA) as a marker of activated GSK-3β and a polyclonal antibody against GSK-3β (dilution 1 : 500, Santa Cruz Biotechnology, Santa Cruz, CA, USA) as a marker of total GSK-3β. Alexa Fluor 488-conjugated goat anti-mouse IgG (dilution 1 : 300) and Alexa Fluor 568-conjugated goat anti-rabbit IgG (dilution 1 : 300) were used as secondary antibodies. Images were captured using a fluorescence microscope system (BX-61/DP70, Olympus). Expression of phosphorylated GSK-3β and total GSK-3β was measured by determining the fluorescence intensity in the cell body of each neuron using a CS analyzer (ATTO). The ratio of GSK-3β (pY216) to GSK-3β (total) was calculated.
3
Histological Analysis of BAT and WAT
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Brown adipose tissue (BAT) and inguinal WAT (ingWAT) samples were fixed in 4% paraformaldehyde (PFA), dehydrated, and embedded in paraffin wax. The paraffin-embedded tissues were cut into 5-μm-thick sections and mounted on slides. The sections were then stained with primary and secondary antibodies according to the manufacturers’ instructions. Data were collected using H&E-stained sections from 4–6 mice in each group (20× and 40× magnifications). UCP1 immunohistochemistry was performed using a polyclonal anti-UCP-1 antibody (1:50). The sections were examined by microscopy (Olympus BX61/DP70). An immunofluorescence analysis of ingWAT was performed using anti-CD137 (1:100) and anti-Ki-67 (1:100) antibodies as well as anti-CD137 (1:100) and anti-UCP1 (1:50) antibodies. All the primary antibodies were incubated overnight at 4 °C. All the sections were incubated with the relevant secondary antibodies (1:250) and DAPI (1:500) for 2 h in the dark at room temperature. All the micrographs were obtained using a TCS SP5 Leica confocal microscope (40× magnification).
4
Amyloid-beta Exposure and Naringenin Treatment
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Embryos were removed from a pregnant ddY mouse (SLC, Shizuoka, Japan) at 14 days of gestation as described previously (Tohda et al., 2012 (link)). The cells were treated with 10 μM Aβ25-35 or 5 μM Aβ1-42 (Sigma–Aldrich, A4559 or A9810) for 3 days before being treated with naringenin or vehicle (0.1% DMSO) for 4 days. The Aβ25-35 or Aβ1-42 was previously incubated at 37°C for 4 or 7 days for aggregation, respectively. The cells were fixed with 4% paraformaldehyde and immunostained at 4°C for 24 h with a monoclonal antibody against pNF-H (1:500) as an axonal marker and a polyclonal antibody against MAP2 (1:2000, Abcam, Cambridge, United Kingdom, cat # ab32454) as a neuronal marker. Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:200) and Alexa Fluor 594-conjugated goat anti-mouse IgG (1:200) were used as secondary antibodies. Fluorescence images were captured using a fluorescence microscope system (BX61/DP70, Olympus) at 644 μm × 855 μm. The lengths of the pNF-H-positive axons were measured using a MetaMorph analyser (Molecular Devices, Sunnyvale, CA, United States).
5
Cytospin Analysis of Sorted Cells
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Sorted EpCAM+CD44+ cells were washed twice and diluted in 200 µl cold PBS containing 2% FBS. Slides and filters were placed into appropriate slots in a cytospin chamber (Stat Spin; Beckman Coulter, Tokyo, Japan) with the cardboard filters facing the center. In the event of few cells being available, 100 µl cold PBS containing 2% FBS was first placed in each cytospin, which was then spun at 250 × g for 5 min at 25°C to pre-wet the filter, allowing more cells to reach the slide. In addition, correct alignment of the filter/slide interface was ensured. For each sample, 200 µl was added to the appropriate wells of the cytospin, lids were applied and centrifugation was performed at 250 × g for 5 min at 25°C. Subsequently, the filters were removed taking care not to disturb the smears on the slides.
Each slide was examined under a microscope to check cell adherence, morphology and monolayer formation. Slides were dried overnight in a desiccator and evaluated using a transmitted light microscope (BX61/DP70; Olympus, Tokyo, Japan) equipped with an ultraviolet light source and filters. A cytotechnologist at the hospital analyzed the sorted cells with regard to the nuclear to cytoplasmic ratio, the overall cell size and the size of the nucleolus.
Each slide was examined under a microscope to check cell adherence, morphology and monolayer formation. Slides were dried overnight in a desiccator and evaluated using a transmitted light microscope (BX61/DP70; Olympus, Tokyo, Japan) equipped with an ultraviolet light source and filters. A cytotechnologist at the hospital analyzed the sorted cells with regard to the nuclear to cytoplasmic ratio, the overall cell size and the size of the nucleolus.
6
Evaluating Sorted EpCAM+ p75NTR+ Cells
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7
Evaluating Tau Phosphorylation in Neuronal Cultures
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Mouse cortical neurons (E14) were cultured in 8-well chamber slides at a density of 1.45 × 104 cells/cm2 for 3 days. For pretreatment experiments, KKT (10 μg/mL), KPL (1 μM), or vehicle was administered to the cells, and Aβ (25–35) (10 μM) was added 1 h later. At 4 h or 72 h after incubation with Aβ, the cells were fixed. For posttreatment experiments, cells were treated with Aβ (25–35) (10 μM) for 24 h. Then, the medium was replaced with medium lacking Aβ (25–35). KKT (10 μg/mL), KPL (1 μM), or vehicle was included in the replacement medium. At 72 h after drug treatment, the cells were fixed and immunostained with a monoclonal antibody against paired helical filament tau (PHF-tau, AT-8 clone, dilution 1 : 200, Thermo Scientific, Rockford, IL, USA). Alexa Fluor 488-conjugated goat anti-mouse IgG (dilution 1 : 300) was used as the secondary antibody. Images were captured using a fluorescence microscope system (BX-61/DP70, Olympus). The level of tau phosphorylation was measured as the fluorescence intensity in the cell body of each neuron using a CS analyzer (ATTO, Tokyo, Japan).
8
Immunohistochemical Analysis of TXSyn
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Under anesthesia with sodium pentobarbital (80 mg/kg, intraperitoneal), the animals were transcardially perfused with PBS following by with 4% paraformaldehyde (PFA). The skin of the rostral back was isolated, post-fixed with 4% PFA and then immersed in 30% sucrose solution for 2 days. The tissue was then embedded in Tissue-Tek1 O.C.T. Compound (Sakura Fineteck Co., Ltd., Tokyo, Japan) and the frozen samples were sectioned at 16 μm with a cryostat (Leica, Wetzlar, Germany).
The sections were washed with PBS and then treated with Protein Block ® (DAKO Co., Hamburg, Germany) followed by 0.3% Triton X-100 in PBS. The sections were treated with anti-TXSyn antibody (Cayman Chemical) at 4°C overnight. For some experiments, sections were treated with the antibody (Cayman Chemical) pre-incubated with its specific antigen prior to staining. Subsequent to washing with PBS, the preparations were incubated with Alexa Fluor 488-conjugated antirabbit IgG (Life Technologies, Carlsbad, CA, USA) for 1 h at room temperature. Fluorescence signals were observed using a fluorescence microscope (BX-61/DP70, Olympus, Tokyo, Japan). Fluorescent intensity was analyzed with NIH Image software (National Institute of Health, Bethesda, MD, USA).
The sections were washed with PBS and then treated with Protein Block ® (DAKO Co., Hamburg, Germany) followed by 0.3% Triton X-100 in PBS. The sections were treated with anti-TXSyn antibody (Cayman Chemical) at 4°C overnight. For some experiments, sections were treated with the antibody (Cayman Chemical) pre-incubated with its specific antigen prior to staining. Subsequent to washing with PBS, the preparations were incubated with Alexa Fluor 488-conjugated antirabbit IgG (Life Technologies, Carlsbad, CA, USA) for 1 h at room temperature. Fluorescence signals were observed using a fluorescence microscope (BX-61/DP70, Olympus, Tokyo, Japan). Fluorescent intensity was analyzed with NIH Image software (National Institute of Health, Bethesda, MD, USA).
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