Relative cell numbers were assessed using a premixed water-soluble tetrazolium salt (WST-1) cell viability test (Takara, Shiga, Japan) according to the manufacturer’s instructions. The cells were seeded at a density of 1 × 104 cells per well. WST-1 was added to each well, and the absorbance of the microplate at 450 nm was measured after an additional 4 h incubation. The data represent three independent experiments (n = 3). DNA-synthesizing cells were visualized using an Ethynyl deoxyuridine (EdU) kit (Invitrogen, CA, USA) following the manufacturer’s instructions. Then, the cells were washed with phosphate-buffered saline, mounted with a 4’, 6-diamidino-2-phenylindole (DAPI)-containing mounting solution (Vectashield, Vector Laboratories, Burlingame, CA, USA), and imaged by microscopy (Nikon Eclipse 80i, Tokyo, Japan). The percentage of EdU-positive cells was examined in HCC cell lines using ImageJ (Bethesda, MD, USA) software. The data represent three independent experiments (n = 3).
4 6 diamidino 2 phenylindole dapi containing mounting solution
Manufactured by Vector Laboratories
Sourced in United States
4',6-diamidino-2-phenylindole (DAPI)-containing mounting solution is a laboratory reagent used to stain and visualize DNA in biological samples. It provides a medium for mounting and preserving specimens for fluorescence microscopy.
Automatically generated - may contain errors
Lab products found in correlation
Wst 1 cell viability test, by Takara Bio (1 mentions)
Ethynyl deoxyuridine edu kit, by Thermo Fisher Scientific (1 mentions)
Eclipse 80i, by Vector Laboratories (1 mentions)
Donkey anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Eclipse 80i, by Nikon (1 mentions)
Dapi containing mounting solution, by Vector Laboratories (1 mentions)
Neutral buffered formalin, by Merck Group (1 mentions)
Objective lens, by Zeiss (1 mentions)
Lsm 510 confocal microscope, by Zeiss (1 mentions)
Alexa fluor 488 conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Alexa fluor 488 or 594, by Thermo Fisher Scientific (1 mentions)
Nestin, by Thermo Fisher Scientific (1 mentions)
Glial fibrillary acidic protein (gfap), by Agilent Technologies (1 mentions)
Axio imager z1, by Zeiss (1 mentions)
A1 spectral confocal microscope, by Nikon (1 mentions)
8 protocols using 4 6 diamidino 2 phenylindole dapi containing mounting solution
1
Cell Viability and Proliferation Assessment
2
Immunofluorescence Assay for IFITM1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cells were seeded onto coverslips in 4-well plates. After 24 h, the cells were treated with either LPS, IRF1 siRNA, or IFITM1 siRNA; washed with PBS; and then fixed with 4% paraformaldehyde. Next, the cells were treated with cold methanol for 5 min and blocking solution (5% BSA in PBS) for 1 h. The cells were then incubated with the primary antibody anti-rabbit IFITM1 (1:200, Abcam, Cambridge, UK) and the secondary antibody donkey anti-rabbit IgG (Jackson Laboratory, West Grove, PA). Thereafter, the cells were washed with PBS, mounted with a 4′,6-diamidino-2-phenylindole (DAPI)-containing mounting solution (Vectashield, Vector Laboratories, Burlingame, CA), and imaged by microscopy (Nikon Eclipse 80i Tokyo, Japan).
3
Immunofluorescence Staining and Peptide Internalization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For staining with anti-NPM, cultured cells were fixed in 10% neutral buffered formalin (Sigma) for 10 min, and then in methanol for 10 min, washed in PBS and incubated with blocking buffer (PBS containing 0.5% bovine serum albumin) for 1 hour prior to incubation with a mouse anti-NPM antibody (Invitrogen) in blocking buffer for overnight at 4°C. Cells were then incubated with Alexa Fluor 488-conjugated secondary antibody (Jackson Immuno Research Laboratories) for 1 hour, washed three times with PBS and counterstained with 4′,6-diamidino-2-phenylindole (DAPI) -containing mounting solution (Vectashield). Stained cells were examined with a LSM 510 confocal microscope using a 63 × objective lens (Zeiss).
To determine the ability of FITC-labeled peptides to enter the cells and to visualize intracellular distribution of the peptides, MCF7 cells were plated on 4-chamber glass cover slides (Lab-Tek) to adhere overnight, incubated with FITC-labeled peptides (30 μM) for 30 min, and then washed three times with PBS before being fixed and mounted with DAPI-containing mounting solution (Vectashield). Images were acquired using a Nikon A1R confocal laser scanning microscope equipped with a 60 × oil-immersion objective lens (SBIC-Nikon Imaging Centre).
To determine the ability of FITC-labeled peptides to enter the cells and to visualize intracellular distribution of the peptides, MCF7 cells were plated on 4-chamber glass cover slides (Lab-Tek) to adhere overnight, incubated with FITC-labeled peptides (30 μM) for 30 min, and then washed three times with PBS before being fixed and mounted with DAPI-containing mounting solution (Vectashield). Images were acquired using a Nikon A1R confocal laser scanning microscope equipped with a 60 × oil-immersion objective lens (SBIC-Nikon Imaging Centre).
4
Immunofluorescence Staining of Neural Stem Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell samples were fixed in 4% paraformaldehyde for 15 min at RT and washed
with phosphate buffed saline (PBS). After permeabilization with 0.05%
Triton X-100 in PBS for 5 min, the samples were blocked using 2% bovine
serum albumin solution for at least one hour and then incubated with primary
antibodies (see below) overnight at 4°C. After that, the samples were
thoroughly washed with PBS thrice and then incubated with appropriate secondary
antibodies conjugated with a fluorescent dye (Alexa fluor® 488
or 594, Thermo Fisher Scientific) for 30 min at RT. After washing with PBS, the
coverslips were mounted on glass slides using a 4’,6-diamidino-
2-phenylindole (DAPI)-containing mounting solution (Vector laboratory,
Burlingame, CA, USA). The cell images were captured using a fluorescence
microscope (IX71) equipped with a digital camera (DP71) (both from Olympus,
Tokyo, Japan). Primary antibodies used in this study were as follows: SOX1
(rabbit, 1:200; R&D Systems, San Diego, CA, USA), SOX2 (rabbit, 1:200;
Thermo Fisher Scientific), NESTIN (Mouse, 1:200; Thermo Fisher Scientific), TUJ1
(Mouse, 1:1,000; BioLegend, San Diego, CA, USA), GFAP (Rabbit, 1:1,000; Dako,
Carpinteria, CA, USA), A2B5 (Mouse, 1:200; Thermo Fisher Scientific), and NG2
(Rabbit, 1:100; Thermo Fisher Scientific).
with phosphate buffed saline (PBS). After permeabilization with 0.05%
Triton X-100 in PBS for 5 min, the samples were blocked using 2% bovine
serum albumin solution for at least one hour and then incubated with primary
antibodies (see below) overnight at 4°C. After that, the samples were
thoroughly washed with PBS thrice and then incubated with appropriate secondary
antibodies conjugated with a fluorescent dye (Alexa fluor® 488
or 594, Thermo Fisher Scientific) for 30 min at RT. After washing with PBS, the
coverslips were mounted on glass slides using a 4’,6-diamidino-
2-phenylindole (DAPI)-containing mounting solution (Vector laboratory,
Burlingame, CA, USA). The cell images were captured using a fluorescence
microscope (IX71) equipped with a digital camera (DP71) (both from Olympus,
Tokyo, Japan). Primary antibodies used in this study were as follows: SOX1
(rabbit, 1:200; R&D Systems, San Diego, CA, USA), SOX2 (rabbit, 1:200;
Thermo Fisher Scientific), NESTIN (Mouse, 1:200; Thermo Fisher Scientific), TUJ1
(Mouse, 1:1,000; BioLegend, San Diego, CA, USA), GFAP (Rabbit, 1:1,000; Dako,
Carpinteria, CA, USA), A2B5 (Mouse, 1:200; Thermo Fisher Scientific), and NG2
(Rabbit, 1:100; Thermo Fisher Scientific).
5
Tirabrutinib Modulates Neutrophil Responses
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Neutrophils isolated from the peripheral blood of healthy volunteers using Polymorphprep were resuspended in RPMI 1640 supplemented with 10% FBS and reacted with tirabrutinib at concentrations of 0, 2.5, 5.0, 10, 25, 50, and 100 nM for 1 h at 37 °C. Neutrophils were applied to eight-well chambers on MPO and anti-MPO-IC-immobilized and IC-nonimmobilized slides (1 × 105/well) and allowed to settle at 37 °C. Four hours later, the samples were fixed with 4% paraformaldehyde (PFA) for 15 min at RT. After removing the chambers from the slides, the samples were mounted with a 4′,6-diamidino-2-phenylindole (DAPI)-containing mounting solution (Vector Laboratories, Newark, CA, USA).
In other experiments, neutrophils pretreated with tirabrutinib were primed with 5 ng/ml TNF-α for 30 min at 37 °C. Cells were applied to eight-well chambers on MPO and anti-MPO-IC-immobilized and IC-nonimmobilized slides (1 × 105/well) and allowed to settle at 37 °C. Four hours later, cells were collected and subjected to FCM to evaluate cell swelling that precedes NET formation [12 (link)]. The time course protocol was determined according to the previous study [13 (link)].
In other experiments, neutrophils pretreated with tirabrutinib were primed with 5 ng/ml TNF-α for 30 min at 37 °C. Cells were applied to eight-well chambers on MPO and anti-MPO-IC-immobilized and IC-nonimmobilized slides (1 × 105/well) and allowed to settle at 37 °C. Four hours later, cells were collected and subjected to FCM to evaluate cell swelling that precedes NET formation [12 (link)]. The time course protocol was determined according to the previous study [13 (link)].
6
Oleate-Induced Lipid Droplet Analysis
7
Quantification of 5-HT and ChA Positive Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Paraffin-embedded tissue sections were deparaffinized and subjected to antigen retrieval for 10 min in 10 mM sodium citrate, pH 6.0 and incubated overnight at 4 °C with primary antibodies (see Supplementary Table 4 ). At the following day the slides were incubated for 2 h at room temperature (RT) with secondary antibodies (see Supplementary Table 4 ) and mounted with 4′,6-diamidino-2-phenylindole (DAPI) -containing mounting solution (Vector Laboratories, Lörrach, Germany) for counterstaining of nuclei. Samples were imaged with an Axio Imager Z1 (Carl Zeiss, Jena, Germany) and 5-HT and ChA positively-stained cells were scored blindly, normalized to total cell number using ImageJ software (NIH, Bethesda, ML, USA) and presented as percentage of total cell number (cells/nuclei (%)).
8
Breast Cancer Cell RUNX2 Expression
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!