Condensation of nuclear chromatin is usually the late apoptotic event and is detected by staining Hoechst 3325843 (link). PC-3 cells containing 2.0 × 105 cells/well were cultured on coverslips, and kept in six-well plates for 12 h. After 72 h treatment with 1i and VP-16, Hoechst 33258 staining was carried out according to the kit’s procedure (Beyotime Institute Biotechnology, China). The cells were viewed under a fluorescence microscopy (Olympus BX53 + DP72) with a ×20 objective lens.
Bx53 dp72
Manufactured by Olympus
Sourced in Japan
The BX53 + DP72 is a microscope system designed for laboratory use. The BX53 is the microscope body, and the DP72 is the digital camera component. The system provides high-quality imaging capabilities for various scientific applications.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33258 staining, by Beyotime (1 mentions)
Dab kit, by Maixin Group (1 mentions)
Cell light edu apollo 488 in vitro imaging kit, by RiboBio (1 mentions)
Image analysis software, by Olympus (1 mentions)
Edu incorporation assay kit, by RiboBio (1 mentions)
U fbw, by Olympus (1 mentions)
Alcian blue, by Olympus (1 mentions)
Ab92486, by Abcam (1 mentions)
Protein block serum free, by Agilent Technologies (1 mentions)
Alexa fluor 594 conjugated donkey anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Ab46154, by Abcam (1 mentions)
Alexa fluor 488 conjugated donkey anti rat igg, by Thermo Fisher Scientific (1 mentions)
Biozero bz 9000 series, by Keyence (1 mentions)
Ab39250, by Abcam (1 mentions)
8 protocols using bx53 dp72
1
Hoechst 33258 Staining for Late Apoptosis
2
IGFBP1 Protein Expression in Xenografts
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Immunohistochemical assays were conducted to determine the IGFBP1 protein expression in xenografted tumors (control and β-elemene-treated groups). Serial (4 mm) sections from paraffin-embedded conventional tissues were deparaffinized in xylene and hydrated in a series of graded alcohols. The antigen was retrieved using a citric acid buffer (pH 6.0) and immersed in 3% H2O2 to inhibit endogenous peroxidase activity followed by incubation in 5% bovine serum albumin to block nonspecific binding. Overnight incubation at 4 °C with a primary antibody against IGFBP1 (dilutions of 1:100, Abcam, Cambridge, UK) was performed followed by incubation with a secondary antibody (Maixin Biotech. Co., Ltd, Fuzhou, China) for 30 min. Detection was conducted using 3,3′-diaminobenzidine in accordance with the instructions provided by the manufacturer (DAB Kit, Maixin Biotech. Co., Ltd., Fuzhou, China). Pictures were taken with a microscope (×200 magnification, Olympus Corporation BX53 + DP72, Tokyo, Japan).
3
Cell Proliferation Assay with EDU
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Cell proliferation was measured by Cell‐Light EDU Apollo 488 In Vitro Imaging Kit (RiboBio) according to instructions from the manufacturer. First of all, HepG2 and Huh‐7 cells in 96‐well plates were treated with SM for 24 h, then incubated with EDU reagent for 2 h and fixed in 4% paraformaldehyde for 30 min. After washing by glycine, cells were incubated in 0.2% Trion X‐100 for 10 min followed by adding 1× Apollo reaction buffer, and then cells were stained with Hoechst (5 mg/ml). Microscope (BX53 + DP72, Olympus Corporation) was used to take images which were evaluated by an image analysis software (Media Cybernetics, Inc.). Percent cell proliferation was calculated as: (EDU positive cells/Hoechst stained cells) × 100.
4
Cell Proliferation Assay Protocol
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Ctrl‐Kyse150 and 12‐LOX‐Kyse150 cells were plated into 24‐well plates (4 × 104 cells/well) for cell climbing. Following adherence of the cells, they were treated with LY294002 or RAD001 for 48 hours in RPMI‑1640 with 10% FBS and then stained with EdU using EdU incorporation assay kit (Ribobio; Guangzhou, China) according to the manufacturer's instructions. Five fields from each cell climbing well were randomly captured with Olympus BX53 DP72, and three independent experiments were performed. The number of EdU‐positive cells was calculated with ImageJ 1.47V.
5
Microscopy Methods for Extracellular and Intracellular Polysaccharides
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To detect extracellular polysaccharides, a 100-μL aliquot of the cell culture, harvested from the main cultivation, was suspended in 20 μL India ink (Daiso Co., Ltd., Japan) and observed under an optical microscope (BX53: Olympus, Tokyo, Japan) at DIC (differential interference contrast) conditions with a shutter speed of 0.48 s. Alcian blue (#015-13805: Wako Co., Ltd., Osaka, Japan) staining was conducted for detecting acidic polysaccharides [[21] , [22] (link), [23] (link)]. PAS (#164-19705: Wako Co., Ltd.) staining was performed for detecting neutral polysaccharides [24 ]. Cells stained with Alcian blue or PAS were observed using BX53 (Olympus) at DIC conditions with a shutter speed of 0.48 s. Regarding intracellular lipid staining [25 (link)], a 1-mL aliquot of the cell culture was collected from the main cultivation and mixed with 20 μM of Nile red (#144-08811: Wako Co., Ltd.). Samples were observed for morphology using an optical photomicroscope at a high resolution with a Nomarski prism or using the fluorescence microscope, BX53/DP72, fitted with a special filter, U-FBW (Olympus) for excitation (460–495 nm) and emission (510 nm). The exposure time for photography was 0.2 s.
6
Histological Analysis of Gastric Ulcer Markers
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Tissue samples encompassing the gastric ulcer area were immediately fixed in 10% neutral buffered paraformaldehyde, dehydrated with a graded series of ethanol solutions, and embedded in paraffin. The paraffin‐embedded blocks were sliced into 3‐μm sections, and each section was mounted onto a glass slide. The sections were activated using 0.01 M citrate buffer (pH 6), preincubated with Protein Block Serum‐Free (Dako), and incubated overnight at 4°C with (a) primary anti‐CD41 antibody (1:50, rat monoclonal, sc‐19963, Santa Cruz Biotechnology), (b) primary anti‐TGF‐β1 antibody (1:200, rabbit polyclonal, ab92486, Abcam) and/or (c) primary anti‐VEGFA antibody (1:50, rabbit polyclonal, ab46154, Abcam), and washed three times with phosphate‐buffered saline (PBS). Sections that had been incubated with anti‐CD41 and anti‐TGF‐β1 or anti‐CD41 and anti‐VEGFA antibody were incubated for 1 h at room temperature with Alexa Fluor 488‐conjugated donkey anti‐rat IgG (1:500, Molecular Probes) and Alexa Fluor 594‐conjugated donkey anti‐rabbit IgG (1:500, Molecular Probes). The samples were imaged with an optical photomicroscope BX53/DP72 (Olympus, Tokyo, Japan) and a fluorescence microscope (Biozero BZ‐9000 Series; Keyence).
7
Cyanobacteria Microscopic Observation
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8
Immunohistochemical Analysis of Xenograft Tumors
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Subcutaneous xenograft tumor tissue of nude mice was fixed with 4% formaldehyde for about 24 h before paraffin-embedding, slicing. After antigen retrieval with microwave method, the primary antibody of PCNA (13110S, diluted into 1:4000, CST), VEGFA (ab39250, diluted into 1:100, Abcam), SP1 (9389S, diluted into 1:2000, CST), CD31 (3528S, diluted into 1:1000, CST) were all incubated at 4°C overnight, followed by secondary antibody for 1 h. 3,3ʹ- diaminobenzidine (Maixin Biotech, Fuzhou, China) was used as chromogenic agent. Pictures were randomly observed and taken under ×400 magnification from at least five random fields by using upright microscope (BX53+DP72, Olympus Corporation, Japan).
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