TUNEL assay was conducted using the DeadEndTM colorimetric TUNEL System (Promega, Madison, WI, USA) according to the manufacturer’s protocol [24 (link)]. The slides were viewed under bright-field inverted microscope (Nikon, Tokyo, Japan).
Bright field inverted microscope
Manufactured by Nikon
Sourced in Japan
The Bright-field inverted microscope is a versatile laboratory instrument designed for the observation of samples. It features an inverted optical design, where the objective lens is positioned below the specimen stage, allowing for the examination of large or opaque samples. The microscope uses bright-field illumination, which provides a high-contrast image by directly illuminating the specimen from below.
Automatically generated - may contain errors
Lab products found in correlation
Deadend colorimetric tunel system, by Promega (3 mentions)
Matrigel, by BD (2 mentions)
Bright field microscope, by Nikon (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Cayman Chemical (1 mentions)
Crystal dissolving solution, by Cayman Chemical (1 mentions)
Rx100 mk2, by Sony (1 mentions)
Matrigel, by Corning (1 mentions)
96 well tissue culture plate, by Thermo Fisher Scientific (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
13 protocols using bright field inverted microscope
1
TUNEL Assay for Apoptosis Detection
2
Apoptosis Detection in Tumor Samples
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The terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labelling assay was performed using a DeadEnd colourimetric apoptosis detection kit (Promega, Wisconsin, USA), according to the manufacturer’s protocol. In brief, tumours were excised and embedded on a slide. Then, the slides were deparaffinised in xylene twice for 5 min, followed by rehydration in decreasing concentrations of ethanol (100, 95, 85, 70 and 50%). Next, the slides were washed in PBS. To detect apoptotic cells, the slides were fixed in 4% paraformaldehyde and permeabilised using proteinase K and then fixed again in 4% paraformaldehyde. The slides were then equilibrated using the equilibration buffer and labelled using TdT. Next, the slides were blocked in hydrogen peroxide before being incubated with streptavidin HRP. The slides were then developed using 3,3′-diaminobenzidine, mounted in glycerol and viewed under a bright-field inverted microscope (Nikon, Japan). The degree of DNA fragmentation was measured based on the presence of dark brown cells (apoptotic cell indicator) against a light brown background.
3
Hypoxia-Induced HUVEC Wound Healing Assay
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Briefly, 5 × 105 hyperglycemia-induced HUVECs were seeded in 6-well plates under hypoxic conditions. Then, scratches were generated using a 200 μL plastic pipette tip when cells were grown to a confluent monolayer, and the cells were then washed with a medium containing 0.5% FBS three times. After treatment with compounds in hypoxia, images of the wounded monolayer of HUVECs were taken at 0, 12, 24, and 36 h using a bright-field inverted microscope (Nikon, Japan).
4
Analyzing Tumor Mitotic Activity
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The harvested tumors were fixed in 10% formalin and were embedded in paraffin before being sliced into thin sections. Then the paraffin sections were stained with hematoxylin and eosin (H&E) and were viewed under a bright-field microscope (Nikon, Japan). The mitotic cells present were counted and compared between the groups. The slides were viewed under a bright-field inverted microscope, and the mitotic cells were counted in at least five fields of the slides from random sections of the tumors (Nikon, Japan).
5
Cytotoxicity Evaluation of Compounds
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The cells were seeded at a density of 1 × 104 in 96-well plates. Following overnight culture, the cells were treated with increasing doses of compounds 1 –3 (i.e., 10, 25, 50, or 100 μM) and incubated for 24 h. Next, 10 µL of MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) reagent (Cayman) was added to each well and after 3 h of incubation (37 °C, 5% CO2) the medium was aspirated, and the formazan produced in the cells appeared as dark crystals at the bottom of the wells. Subsequently, crystal dissolving solution (Cayman) was added to each well and the optical density (OD) was determined at 570 nm on a plate reader (BIOTEK). Doxorubicin (DOX) was used as a positive control. When hepatotoxicity of test compounds was evaluated, the morphology of the cells was also determined using a bright-field inverted microscope (Nikon).
6
Trypan Blue Cell Viability Assay
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Cells were incubated with 100 µL of trypan Blue 0.4% solution (Invitrogen, Carlsbad, CA, USA) for 15 min, washed twice with PBS 1X and imaged in bright field inverted microscope (Nikon TMS, Tokyo, Japan). Pictures were taken using a Digital Camera (Sony RX100 MK2, Japan).
7
Colorimetric TUNEL Assay for Apoptosis Detection
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The TUNEL assay was carried out using the DeadEnd™ colorimetric TUNEL System (G7360, Promega, USA) according to the manufacturer’s protocol. In brief, tumors were excised and embedded on slide. Then, the slides were deparaffinized in xylene twice for 5 min before they were rehydrated in decreasing concentrations of ethanol (100, 95, 85, 70 and 50%) beforethey were washed in PBS. To detect apoptotic cells, the slides were fixed in 4% paraformaldehyde and permeabilized using proteinase K before they were fixed again in 4% paraformaldehyde. Later, the slides were then equilibrated using the equilibration buffer and labeled using terminal deoxynucleotidyl transferase (TdT). Next, the slides were blocked in hydrogen peroxide before being incubated with streptavidin horseradish peroxidase (HRP). The slides were then developed using 3,3′-Diaminobenzidine (DAB) and mounted in glycerol and they were viewed under a bright-field inverted microscope (Nikon, Japan). The degree of DNA fragmentation was measured based on the presence of dark brown cells (apoptotic cell indicator) against a light brown background.
8
Histological Analysis of Organ Sections
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The sections of organs and tumor immersed in 10% formalin were paraffin-embedded, stained with haematoxylin and eosin and viewed under a bright-field inverted microscope (Nikon, Japan). Histological architecture and mitotic cells between groups were compared.
9
Quantifying Apoptosis in Tumor Sections
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The degree of apoptosis induction of treatments was determined using the DeadEnd™ colorimetric TUNEL System (Promega, USA) according to the manufacturer’s protocol. The paraffin was removed from the embedded tumor-sectioned slides by immersing them into xylene, followed by rehydration using graded ethanol (100%, 95%, 85%, 70% and 50%), and fixation using 4% paraformaldehyde. Tissue sections were subsequently permeabilized using Proteinase K and equilibrated using equilibration buffer. Fragmented DNA was labelled by incubation with rTdT mixture and 2x SSC termination solvent was used to terminate the reaction. The slides were then, incubated with streptavidin HRP followed by incubation with substrate solution (DAB) for colorimetric detection. The slides were mounted with glycerol and examined under bright-field inverted microscope (Nikon, Japan) (Ben-Izhak et al., 2007 (link)).
10
Antiangiogenic Effects of Zileuton
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To identify the antiangiogenic effect of zileuton, tube formation assay was conducted on Matrigel (BD Biosciences, Bedford, MA, USA)-coated plates as previously reported [32 (link)]. Firstly, the growth factor-reduced Matrigel was melted at 4 °C and then plated 300 µL/well Matrigel into 24-well plates. The Matrigel-coated plate was placed on ice at a horizontal level for 10 min to distribute Matrigel evenly and then incubated at 37 °C for 30 min polymerization. Then, cells (1 × 105 cells/well) were seeded on the Matrigel-coated plate and pretreated with or without IBTX (0.3 µM, BK channel blocker) for 30 min. After pretreatment of IBTX, cells were treated with either zileuton (1–50 µM) or NS1619 (10 µM) for 1 h and then followed by the addition of VEGF (10 ng/mL) for indicated time points (4–6 h after VEGF stimulation) with 5% CO2 at 37 °C. Images and quantification of endothelial network formation was performed by counting the number of tubes formed per field in each well using an inverted (bright-field) microscope (Nikon) to evaluate the antiangiogenic capacity of zileuton.
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