Daudi cells were adsorbed on polyLysine-treated slides and fixed with Formalin (Sigma) containing 1% Triton-X-100. Cells were labeled with anti-Vpr antibody followed by labeling with a mixture of Alexa488-conjugated donkey anti-goat and anti-rabbit IgG (H + L) antibodies (Life Technologies). Nuclei were stained with DAPI staining solution from Sigma Aldrich. Cells were mounted on glass slides covered with anti-fade medium (Hardset Vectashield). Two-color images were obtained with a light microscope, Leica DC250 (Leica) with a Plan Apo 63 ×/1.32–0.6 oil-immersion objective lens. Digital images were processed with the ImageJ software (NIH Image). Percentages of Vpr+ Daudi B-cells were determined by using the Cell Counting plugin of ImageJ. A minimum of 750 cells were counted per 6-well plate.
Dapi staining solution
Manufactured by Merck Group
Sourced in United States
DAPI staining solution is a fluorescent dye used in microscopy and flow cytometry applications. It binds to the minor groove of DNA, allowing for the visualization and quantification of nucleic acids in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 780 confocal microscope, by Zeiss (3 mentions)
Formalin, by Merck Group (2 mentions)
Alexa488 conjugated donkey anti goat, by Thermo Fisher Scientific (2 mentions)
Anti rabbit igg h l antibodies, by Thermo Fisher Scientific (2 mentions)
Dc250, by Leica (2 mentions)
Cm3050, by Leica (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Te2000 u, by Nikon (1 mentions)
Imagexpress micro xl, by Molecular Devices (1 mentions)
Metaxpress, by Molecular Devices (1 mentions)
Multidrop combi liquid handler, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 phalloidin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dmem medium, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Ros assay kit, by Beyotime (1 mentions)
Cck 8 kit, by Beyotime (1 mentions)
Bca protein concentration assay kit, by Beyotime (1 mentions)
Alexa fluor 555 conjugated donkey anti mouse igg, by Thermo Fisher Scientific (1 mentions)
10 protocols using dapi staining solution
1
Immunofluorescent Detection of Vpr in Daudi Cells
2
Immunohistochemical Analysis of TGF-β
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Paraffin sections were deparaffinised, antigen retrieved, blocked, and then incubated overnight at 4°C with TGF‐β primary antibody (1:300) and horseradish peroxidase‐labelled secondary antibody. All the above antibodies were purchased from Abcam. Nuclear staining was performed in the dark by adding DAPI staining solution (Sigma, USA), and sections were sealed with anti‐fluorescence quenching sealant. Sections were observed under a fluorescence microscope (TE2000‐U, Nikon, Japan), and images were collected.
3
Immunocytochemistry of Adipocytes
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Primary brown and white adipocytes grown on coverslips were fixed with 4% paraformaldehyde and then permeabilized by Triton X-100 (0.1%) in phosphate-buffered saline (PBS) for 10 min. The cells were then blocked with 5% bovine serum albumin (BSA) in PBS for 30 min and stained with first antibodies (anti-NICD or Flag) overnight. After washing, the fixed cells were then stained with an anti-rabbit Alexa Fluor® 568 antibody (1:400) or an anti-mouse Alexa Fluor® 488 antibody (1:400) for 60 min. Cell nuclear DNAs were detected by DAPI Staining Solution (Sigma). Confocal images were taken by a Zeiss LSM 780 confocal microscope.
4
High-Content Screening of Antitrypanosomal Compounds
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Samples
and pure compounds
from the fungal natural product library, benznidazole (Sigma cat.
no. 419656), and DMSO (Sigma cat. no. D2650) were spotted onto black,
clear-bottom 384-well plates (Greiner Bio One, 782092) using an Acoustic
Transfer System (ATS) instrument (EDC Biosystems). Using a Multidrop
Combi liquid handler (Thermo Scientific), 500 cells per well of C2C12
cardiomyoblasts and 7500 cells per well of CA-I/72 T. cruzi parasites were added to each well plate.
This was followed by incubation at 37 °C and 5% CO2 for 72 h using plate-holding trays to reduce the evaporation of
cultures at the edges of the plates. Paraformaldehyde (4% final concentration)
in 1× phosphate-buffered saline (Invitrogen, 10010023) was then
added to the plates to fix the cells. Subsequent treatment with 5
μg/mL DAPI staining solution (Sigma-Aldrich, D9542) was applied
for 1 h to stain host cells and parasite nuclei. Imaging of well plates
was conducted with a 10× fluorescence objective using an ImageXpress
Micro XLS automated high-content imager (Molecular Devices). A custom
image analysis module generated in MetaXpress (Molecular Devices)
was then used to count host cells and parasite nuclei to be used as
an assay readout, as previously described.23 (link) Microscopic figure images were prepared using ImageJ.39 (link)
and pure compounds
from the fungal natural product library, benznidazole (Sigma cat.
no. 419656), and DMSO (Sigma cat. no. D2650) were spotted onto black,
clear-bottom 384-well plates (Greiner Bio One, 782092) using an Acoustic
Transfer System (ATS) instrument (EDC Biosystems). Using a Multidrop
Combi liquid handler (Thermo Scientific), 500 cells per well of C2C12
cardiomyoblasts and 7500 cells per well of CA-I/72 T. cruzi parasites were added to each well plate.
This was followed by incubation at 37 °C and 5% CO2 for 72 h using plate-holding trays to reduce the evaporation of
cultures at the edges of the plates. Paraformaldehyde (4% final concentration)
in 1× phosphate-buffered saline (Invitrogen, 10010023) was then
added to the plates to fix the cells. Subsequent treatment with 5
μg/mL DAPI staining solution (Sigma-Aldrich, D9542) was applied
for 1 h to stain host cells and parasite nuclei. Imaging of well plates
was conducted with a 10× fluorescence objective using an ImageXpress
Micro XLS automated high-content imager (Molecular Devices). A custom
image analysis module generated in MetaXpress (Molecular Devices)
was then used to count host cells and parasite nuclei to be used as
an assay readout, as previously described.23 (link) Microscopic figure images were prepared using ImageJ.39 (link)
5
PLGA Nanoparticles for Doxorubicin Delivery
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CUR (≥94%), PFOB, polyvinyl alcohol (PVA), DAPI staining solution, TritonX-100 were purchased from Sigma-Aldrich (MO, USA); PLGA (50:50, molecular weight [MW] = 12,000) was purchased from Daigang Biomaterials Co., Ltd(Jinan, China); DOX hydrochloride (97%) was purchased from Aladdin; dichloromethane (DCM), isopropanol and triethylamine were purchased from Chemical Book (Beijing, China); DMEM medium and fetal bovine serum (FBS) were purchased from Gibco (MA, USA); Live/Dead cell kit and Alexa Fluor 594 phalloidin were purchased from Thermo Fisher; BCA protein concentration assay kit and ROS assay kit were purchased from Beyotime; CCK-8 kit and antibodies for AKT, pAKT, cleaved Caspase-3, BAX, BCL-2, HIF-1α and β-actin were purchased from Abcam (Cambridge, UK).
6
Visualizing TAZ Localization in Transfected HEK293T
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HEK293T cells transfected with indicated plasmids were plated on coverslip and incubated at 37 °C for 48 h. Then cells were fixed in 4% paraformaldehyde for 20 min, washed three times in PBS and permeabilized with 0.5% v/v Triton (Cat#T8787, Sigma). After prehybridization at 50 °C for 2 h, 25 nM encoding probes in encoding hybridization buffer was added to the cell-containing coverslip. Samples were incubated in a hybridization oven at 50 °C overnight. Cells were washed with 2× saline-sodium citrate buffer (SSC) twice at 50 °C for 5 min, and incubated with 2×SSC and 50% v/v formamide three times at 50 °C for 25 min. Then samples were washed four times with PBST (0.1% Tween in PBS), once with PBS at room temperature for 5 min, blocked with normal goat serum for 1 h. The samples were incubated with anti-digoxin-fluorescein antibody (1:100, cat# 11207741910, Roche) and mouse anti-human TAZ antibody (1:100, clone: CL0371, cat# ab242313, Abcam) at 4 °C overnight. After that, samples were washed thrice with PBST, incubated with Alexa Fluor 555-conjugated donkey anti-mouse IgG (1:300, cat# A-31570, ThermoFisher Scientific) for 1 h at room temperature and stained with DAPI staining solution (Sigma). Images were taken by laser scanning confocal microscopy (LSM 880, Carl Zeiss).
7
Immunohistochemical Analysis of Brain Sections
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Coronal section of 30 μm was obtained by a cryostat (Leica Microsystems CM 3050S, Wetzlar, Germany) and kept in a cryoprotectant solution at −20°C.
First, free-floating slices were selected and placed on a 24-wells plaque. After that, were washed five times with PBS 0.01M + 1% Triton X-100. Then, free-floating sections were blocked with a solution containing 5% fetal bovine serum (FBS), 1% Triton X-100, PBS 0.01M + gelatine 0.2% for 2h at room temperature. Afterward, slices were washed with PBST (PBS 0.1M, 1% Triton X-100) five times for 5 minutes each and were incubated with the primary antibodies listed inTable 3 , over-night at 4°C. On the following day, coronal slices were washed with PBST 6 times for 5 minutes and then incubated with the secondary antibodies at room temperature for 2h. Later, sections were co-incubated with, 1mg/ml DAPI staining solution (Sigma-Aldrich, St. Louis, MI) for 5 minutes in the dark at room temperature and washed with PBS 0.01M. Finally, the slices were mounted using Fluoromount G (EMS, USA) and image acquisition was performed with a fluorescence laser microscope (Olympus BX51, Germany). At least 3 images from 4 different individuals by the group were analyzed with ImageJ/Fiji software available online from the National Institutes of Health.
First, free-floating slices were selected and placed on a 24-wells plaque. After that, were washed five times with PBS 0.01M + 1% Triton X-100. Then, free-floating sections were blocked with a solution containing 5% fetal bovine serum (FBS), 1% Triton X-100, PBS 0.01M + gelatine 0.2% for 2h at room temperature. Afterward, slices were washed with PBST (PBS 0.1M, 1% Triton X-100) five times for 5 minutes each and were incubated with the primary antibodies listed in
8
Daudi B-Cell Vpr Localization Analysis
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Daudi cells were adsorbed on polyLysine-treated slides and xed with Formalin (Sigma) containing 1% Triton-X-100. Cells were labeled with anti-Vpr antibody followed by labeling with a mixture of Alexa488conjugated donkey anti-goat and anti-rabbit IgG (H+L) antibodies (Life Technologies). Nuclei were stained with DAPI staining solution from Sigma Aldrich. Cells were mounted on glass slides covered with anti-fade medium (Hardset Vectashield). Two-color images were obtained with a light microscope, Leica DC250 (Leica) with a Plan Apo 63 × /1.32-0.6 oil-immersion objective lens. Digital images were processed with the ImageJ software (NIH Image). Percentages of Vpr + Daudi B-cells were determined by using the Cell Counting plugin of ImageJ. A minimum of 750 cells were counted per 6-well plate.
9
Coronal Section Immunofluorescence Staining
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Coronal section of 30 μm was obtained by a cryostat (Leica Microsystems CM 3050S, Wetzlar, Germany) and kept in a cryoprotectant solution at -20°C.
First, free-floating slices were selected and placed on a 24-wells plaque. After that, were washed five times with PBS 0.01M + 1% Triton X-100. Then, free-floating sections were blocked with a solution containing 5% fetal bovine serum (FBS), 1% Triton X-100, PBS 0.01M + gelatine 0.2% for 2h at room temperature. Afterwards, slices were washed with PBST (PBS 0.1M, 1% Triton X-100) five times for 5 min each and were incubated with the primary antibodies over-night at 4°C (Supplementary Table 2). On the following day, coronal slices were washed with PBST 6 times for 5 min and then incubated with the secondary antibodies (Supplementary Table 2) at room temperature for 2h. Later, sections were co-incubated with,1mg/ml DAPI staining solution (Sigma-Aldrich, St. Louis, MI) for 5 min in the dark at room temperature and washed with PBS 0.01M.
Finally, the slices were mounted using Fluoromount G (EMS, USA) and image acquisition was performed with a fluorescence laser microscope (Olympus BX41, Germany). At least four images from 4 different individuals by the group were analysed with ImageJ/Fiji software available online from the National Institutes of Health.
First, free-floating slices were selected and placed on a 24-wells plaque. After that, were washed five times with PBS 0.01M + 1% Triton X-100. Then, free-floating sections were blocked with a solution containing 5% fetal bovine serum (FBS), 1% Triton X-100, PBS 0.01M + gelatine 0.2% for 2h at room temperature. Afterwards, slices were washed with PBST (PBS 0.1M, 1% Triton X-100) five times for 5 min each and were incubated with the primary antibodies over-night at 4°C (Supplementary Table 2). On the following day, coronal slices were washed with PBST 6 times for 5 min and then incubated with the secondary antibodies (Supplementary Table 2) at room temperature for 2h. Later, sections were co-incubated with,1mg/ml DAPI staining solution (Sigma-Aldrich, St. Louis, MI) for 5 min in the dark at room temperature and washed with PBS 0.01M.
Finally, the slices were mounted using Fluoromount G (EMS, USA) and image acquisition was performed with a fluorescence laser microscope (Olympus BX41, Germany). At least four images from 4 different individuals by the group were analysed with ImageJ/Fiji software available online from the National Institutes of Health.
10
Apoptosis Induction by 4-O-Methylhonokiol
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4-O-Methylhonokiol (MH) was generously supplied by Professor Heon Sang Jung (Department of Food Science, Chungbuk National University, Cheongju, Korea). DAPI staining solution and propidium iodide (PI) were obtained from Sigma (St. Louis, MO, USA). The Fluorescein isothiocyanate (FITC) annexin V Apoptosis Detection Kit I was purchased from BD Biosciences (San Diego, CA, USA). Antibodies specific to caspase-3, caspase-9, caspase-8, PTEN, polyADP ribose polymerase (PARP), Bax, Bcl-2, Bcl-XL, and α-mouse IgG horseradish-peroxidase (HRP)-conjugated secondary antibody were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies specific to GAPDH, PPARγ, Akt, p-Akt1/2/3, and anti-goat IgG HRP-conjugated secondary antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The anti-rabbit IgG HRP-conjugated secondary antibody was purchased from Assay Designs (Ann Arbor, MI, USA). JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl benzimidazolylcarbocyanine iodide) was purchased from Enzo (Farmingdale, NY, USA).
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