EdU is a thymidine analog that can be incorporated into replicating DNA for the detection of cell proliferation (Salic and Mitchison, 2008 (link)). In this study, NSCLC cells were seeded into 96-well plates (4 × 103 cells/well) and incubated for 24 h before treatment with different concentrations of SOL (0, 50, 100, or 200 μg/ml) for 24 h. Cells were then incubated for 2 h with 50 μM EdU (#K1075, APE×BIO, TX, United States) before fixation with 4% formaldehyde (#1810473, Biosharp, Hefei, China) for 30 min. EdU Click buffer was then added for 30 min and cells were stained with Hoechst 33342 for 30 min. EdU-positive cells were imaged with an ImageXpress Micro 4 High-Content Screening System (Molecular Devices, CA, United States) at ×100 magnification. Cell proliferation was analyzed as the percentage of viable cells using the following formula:
Formaldehyde
Manufactured by Biosharp
Sourced in China
Formaldehyde is a colorless, flammable gas with a pungent odor. It is commonly used as a preservative and disinfectant in various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Imagexpress micro 4 high content screening system, by Molecular Devices (1 mentions)
Uranyl acetate, by Polysciences (1 mentions)
Sodium cacodylate buffer, by Merck Group (1 mentions)
Lead citrate, by Polysciences (1 mentions)
Spurr s resin, by Polysciences (1 mentions)
7650 tem, by Hitachi (1 mentions)
Osmium tetroxide, by Merck Group (1 mentions)
Glutaraldehyde, by Solarbio (1 mentions)
Confocal dishes, by Solarbio (1 mentions)
Bovine serum albumin (bsa), by Beyotime (1 mentions)
Triton x 100, by Beyotime (1 mentions)
24 well plate, by Thermo Fisher Scientific (1 mentions)
Crystal violet, by Beyotime (1 mentions)
Glycine, by neoFroxx (1 mentions)
Protein a g conjugated agarose beads, by Smart-Lifesciences (1 mentions)
Proteinase k, by Tiangen Biotech (1 mentions)
Tianamp genomic dna kit, by Tiangen Biotech (1 mentions)
Bioruptor plus, by Diagenode (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
12 protocols using formaldehyde
1
EdU-Based Cell Proliferation Assay in NSCLC
2
Ultrastructural Changes in Aspergillus Biofilm
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TEM was performed to investigate the ultrastructural changes of A. fumigatus biofilm after exposure to CRA. In brief, 4 ml A. fumigatus suspension (4×105 conidia) was added to a 25 mm2 cell culture flask with a vented cap, followed by adhesion for 4 h. Subsequently, the supernatant containing non-adherent cells was removed, followed by incubation at 37°C for 24 h after adding fresh RPMI-1640 medium. For the CRA treatment group, CRA was added to fresh RPMI-1640 medium added after 4 h of adhesion. Biofilms were washed in PBS and fixed in a solution of 2.5% glutaraldehyde (Solarbio, Beijing, China) and 4% formaldehyde (Biosharp, Hefei, China) in 0.1 M sodium cacodylate buffer (pH 7.2, Sigma-Aldrich; Merck Millipore). Subsequently, the mixture was fixed for 2 h in the same buffer containing 1% osmium tetroxide (pH 7.2, Sigma-Aldrich, Merck Millipore), and then embedded in Spurr's resin (Polysciences, Warrington, USA). Ultrathin sections were stained with 4% uranyl acetate (Polysciences) and 2.6% lead citrate (Polysciences). Finally, the images were observed under a Hitachi 7650 TEM (Hitachi, Tokyo, Japan) at 80 kV.
3
Immunofluorescence Staining of GC Cells
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GC cells were seeded on confocal dishes (Solarbio, Beijing, China) and incubated in proper media for one night. Then the cells were fixed with formaldehyde (Biosharp, Hefei, Anhui, China) and permeabilized with 0.2% TritonX‐100 (Beyotime, Shanghai, China), which followed by blocking with 1% bovine serum albumin (BSA; Beyotime, Shanghai, China) for 30 min. Next, cells were incubated with the primary antibody overnight at 4°C and secondary antibody for 2 h on the next day. Finally, cells were detected with a confocal microscope (Leica, Weszler, Hesse, Germany). The reagents required above were obtained from Beyotime. The antibodies used in this study are listed in Supplementary Table S1 .
4
Transwell Assay for Cell Migration
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Cells were treated with SBSGL at 0, 600, 1200 and 1800 μg/mL concentrations and incubated for 48 h. Transwell in 24‐well plates (Thermo Fisher Scientific, lot no. 140644) were employed for the transwell assay. Cells in serum‐free medium were added to the upper chamber at a 2.5 × 105 cells/mL density, while the lower chamber contained culture medium. After 48 h of incubation under normal conditions, the chambers were separated and washed with PBS, and the cells on the lower surface were fixed with 4% formaldehyde (Biosharp, lot no. 1912A05) for 15 min. Staining with 700 μL crystal violet (Biyuntian Bio‐Technology Co., lot no. C0121) for 30 min followed. After washing with PBS, cell counting was performed under a microscope.
5
ChIP Assay for Protein-DNA Interactions
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The ChIP assay was performed as described previously (28 (link), 29 (link)). Briefly, 1 × 107/mL cells were fixed in 1% formaldehyde (Biosharp, Hefei, China) for 20 min at room temperature, and 125 mM glycine (BioFroxx, China) was added to quench formaldehyde. After a 30-min incubation in ChIP lysis buffer (50 mM Tris-HCl [pH 8.0], 5 mM EDTA, 150 mM NaCl, 1% NP-40, 0.1% SDS, protease inhibitor) on ice, the lysates were sheared by sonication using a Bioruptor Plus (Diagenode, Liège, Belgium). Cross-linked chromatin samples were incubated with the indicated antibodies or normal rabbit IgG in a rotator at 4°C overnight. Subsequently, protein A/G-conjugated agarose beads (Smart-Lifesciences, Changzhou, China) were added, and the samples were incubated overnight at 4°C in a rotator, collected, and washed three times. To elute DNA fragments, immunocomplexes were incubated with elution buffer (50 mM Tris-HCl [pH 8.0], 10 mM EDTA, 1.0% SDS) for 2 h at 65°C. One half of the eluted immunocomplexes was saved as the immunoprecipitation sample, while the other half was treated with proteinase K (Tiangen Biotech) overnight at 55°C. Finally, the DNA was purified with a TIANamp Genomic DNA kit (Tiangen Biotech). The purified DNA was detected by qPCR. The antibodies used for ChIP and the primers used for qPCR are listed in Tables 3 and 4 .
6
Immunofluorescence Staining of PD-L1 and IFIT1
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Cells were seeded into eight-well chamber slides. After adherence and treatment, cells were fixed using 4% formaldehyde (Biosharp), permeabilized using Triton-X-100 for 10 minutes and blocked with 1% BSA for 1 hour. Following incubated with primary antibodies overnight at 4°C, cells were incubated with fluorescently-labeled conjugated secondary antibodies (Alexa Fluor 594 or 488; Invitrogen). Slides were washed with PBS, stained with DAPI and sealed with coverslips. Fluorescence was observed with Zeiss LSM710 confocal microscope. The antibodies used in the experiment are listed as follow: Rabbit anti-PD-L1 (CAT#17952-1-AP, Proteintech), 1:100 dilution; Mouse anti-IFIT1(CAT#ab118062, abcam), 1:100 dilution. The above experiments were conducted independently three times.
7
Clinicopathological Characterization of HCC
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A total of 33 pairs of HCC tumor and peri-tumor tissue samples were obtained from the Department of Hepatobiliary and Pancreatic Surgery of Zhongnan Hospital of Wuhan University (Wuhan, China). The written informed consent forms were obtained from the patients in this study. Paired HCC tumor tissues and adjacent normal tissues were resected from patients who were clinically and histologically diagnosed HCC without formerly chemotherapy or radiotherapy. Each specimen was split into three parts, and each part was preserved in RNAlater (Invitrogen, Carlsbad, CA, USA), homogenized in RIPA buffer (Biosharp, China) supplemented with 1% phenylmethanesulfonyl fluoride (PMSF, Biosharp, China), protease inhibitors and phosphatase inhibitor cocktail (Biosharp, Anhui, China), and fixed overnight in formaldehyde (Biosharp, China). All patients were regularly followed-up after the operation.
8
Histological Analysis of Liver Tissue
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Small sections of liver tissues were removed from the edges of the left liver lobe (0.5 cm) and fixed with 10% formaldehyde (BioSharp, Hefei, China). Following routine dehydration, transparency and paraffin embedding (Shanghai Yuanye Bio-Technology Co., Ltd., Shanghai, China), the sections (5 μm thick) were stained with hematoxylin and eosin (H&E; Nanjing Jiancheng Bioengineering Institute), and the histomorphology was observed under a light microscope (Olympus CX21; Olympus Corporation, Tokyo, Japan).
9
Immunofluorescence Staining of COUP TF1 and Ki67
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Cells were fixed with 4% formaldehyde (BioSharp, Hefei, China) for 10 min at room temperature. Cells were then permeabilized with 0.5% Triton X-100 (BioSharp, Hefei, China) for 2 min and treated with a blocking serum (Solarbio, Beijing, China) for more than 5 h. Primary antibody, anti-COUP TF1 (sc-74560, 1:50, Santa Cruz, TX, USA), and anti-Ki67 (ab15580, 1:200, Abcam, Cambridge, UK) were incubated overnight for immunofluorescence. After being washed to remove unbound primary antibodies, cells were incubated with the secondary antibody Dnk pAb to Rb IgG (Alexa Fluor® 488) (ab150074, 1:1000, Abcam, UK), donkey anti-mouse IgG H&L (Alexa Fluor 594) (ab15010B, 1:1000, Abcam, UK), and DAPI (Biosharp, Hefei, China) for 2 h under dark conditions. Images were acquired with a Leica SP8 (Wetzlar, Germany) confocal microscope.
10
EdU Proliferation Assay Protocol
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EdU assay was conducted by using EdU assay Kit (Yeasen, China) according to the manufacturer's instructions. In brief, cells were incubated in growth medium containing EdU for 2 h. Then, the cells were fixed in 4% formaldehyde (Biosharp, China) for 20 min and permeabilized with 0.3% TritonX-100 (Sigma, USA) for 15 min. Subsequently, cells were incubated at room temperature for 30 min in the click reaction cocktail. Cell nuclei were stained by Hoechst 33342 for 30 min. Images were captured under confocal microscopy (Carl Zeiss, Germany).
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