WPMY1 cells were seeded on poly‐lysine‐coated coverslips and cultured for 48 h in RPMI 1640 containing 5% FBS. Then, cells were starved overnight and treated with mKLK4, KLK4 (20 nm ) or AP1 (100 μm ) for 48 h. After treatment, cells were fixed and incubated overnight at 4 °C with an anti‐TAGLN or anti‐αSMA antibody, followed by incubation with species‐appropriate secondary antibody coupled with Alexa Fluor® 488 conjugate. Cell nuclei were stained with DAPI before imaging with an Olympus FV1200 laser scanning confocal microscope. For αSMA staining, WPMY1 cells were seeded in 96‐well plates (5000 cells per well) in RPMI 1640 + 5% FBS. After 24 h, cells were treated with mKLK4, KLK4 (20 nm ) or AP1 (100 μm ) over 6 days (treatment renewed every 48 h). Cells were washed and fixed, incubated as above with an anti‐αSMA antibody and then a goat anti‐mouse IgG (H+L) secondary antibody coupled with Alexa Fluor 488 conjugate and nuclei were stained with DAPI. Imaging was performed with an epifluorescent microscope (IX73 Olympus inverted microscope system) and quantitative measurement was taken using the Incucyte live cell imaging system (Essen BioScience, Ann Arbor, MI, USA). Results presented correspond to mean of fluorescence units ± SD obtained in three independent experiments.
Ix73 inverted microscope system
Manufactured by Olympus
Sourced in United States, Japan
The IX73 Inverted Microscope System is a high-performance inverted microscope designed for a variety of applications. It features a sturdy, stable design and advanced optics to provide clear, detailed imaging. The system is equipped with a wide range of accessories and options to accommodate different sample types and imaging requirements.
Automatically generated - may contain errors
Lab products found in correlation
Dp74 color camera, by Olympus (4 mentions)
Sterile 16 well culture slides, by Thermo Fisher Scientific (2 mentions)
Alexa fluor fluorescent secondary antibody, by Thermo Fisher Scientific (2 mentions)
Cellsens imaging software, by Olympus (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Fv1200 laser scanning confocal microscope, by Olympus (1 mentions)
Incucyte live cell imaging system, by Sartorius (1 mentions)
Dp74 colour camera, by Olympus (1 mentions)
Tricaine, by Merck Group (1 mentions)
Prolong diamond antifade mountant with 4 6 diamidino 2 phenylindole, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 conjugated goat anti mouse immunoglobulin g, by Thermo Fisher Scientific (1 mentions)
17 protocols using ix73 inverted microscope system
1
Evaluating KLK4 and AP1 Regulation of Myofibroblast Markers
2
Immunofluorescence Staining of E2-Treated Cells
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Cells were plated on glass coverslips, cultured with serum-free medium overnight, and then stimulated with control or E2 (10 nM) for 24 h. Cells were fixed with 4% paraformaldehyde for 15 min, blocked in 1% BSA/PBS/Tween 20 (0.05%) for 30 min, incubated with primary antibody overnight at 4°C, and then respective secondary antibodies for 1 hour. Negative control for the staining was performed with the omission of primary antibodies. Images were captured using an IX73 Olympus Inverted Microscope System (Olympus).
3
SARS-CoV-2 Nucleocapsid Protein Droplet Formation
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The formation of liquid droplets was imaged on 50 μl of the N protein samples by DIC microscopy (OLYMPUS IX73 Inverted Microscope System with OLYMPUS DP74 Colour Camera) as previously described (Kang et al.,2019 (link)
a; Dang et al.,2021 ). The N protein samples were prepared at 10 μM in 25 mM HEPES buffer (pH 7.0) with 70 mM KCl (buffer 1), while NTD and CTD samples were prepared at 100 and 200 μM, respectively in 10 mM sodium phosphate buffer (pH 7.0) in the presence of 150 mM NaCl (buffer 2) for NMR studies. HCQ at 10 mM was dissolved in buffer 1 for LLPS or buffer 2 for NMR binding studies with the final pH adjusted to 7.0.
a; Dang et al.,2021 ). The N protein samples were prepared at 10 μM in 25 mM HEPES buffer (pH 7.0) with 70 mM KCl (buffer 1), while NTD and CTD samples were prepared at 100 and 200 μM, respectively in 10 mM sodium phosphate buffer (pH 7.0) in the presence of 150 mM NaCl (buffer 2) for NMR studies. HCQ at 10 mM was dissolved in buffer 1 for LLPS or buffer 2 for NMR binding studies with the final pH adjusted to 7.0.
4
Imaging Liquid Droplet Formation of N Protein
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The formation of liquid droplets was imaged on 50 μl of the N protein samples by DIC microscopy (OLYMPUS IX73 Inverted Microscope System with OLYMPUS DP74 Color Camera) as previously described.4 , 14 , 19 The full‐length N protein samples were prepared at 10 μM in 25 mM HEPES buffer (pH 7.0) with 70 mM KCl. The turbidity (absorption at 600 nm) were measured for all DIC samples with three repeats.
5
Paraffin Embedding and Histological Analysis of Zebrafish
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Six‐month‐old adult zebrafish were sacrificed by cold 4 mg/ml of N‐(Tris[hydroxymethyl]methyl) glycine (Tricaine, Merck, Kenilworth, NJ, USA) and then immediately fixed for 1 week with Bouin solution (75% saturated picric acid, 10% formaldehyde, 5% glacial acetic acid). After fixation, zebrafish were transferred to 70% ethanol to remove excess picric acid. For paraffin embedding, zebrafish were gradually dehydrated as follows: 70% ethanol for 1 hr, 80% ethanol for 1 hr, 90% ethanol for 1 hr, and twice absolute ethanol for 1 hr. Then, zebrafish were incubated at 60°C and infiltrated as follows: first, cleared by xylene for 15 min; second, cleared by 50% xylene and 50% soft paraffin for 15 min; and finally, infiltrated by hard paraffin for 25–30 min. Lastly, zebrafish were immediately embedded in hard paraffin and then properly trimmed to cutting size. Paraffin blocks were then sectioned at 7 μm by microtome. Histological hematoxylin–eosin (H&E) staining of the sections was subsequently performed using standard protocols. Sections were mounted with Fisher ChemicalTM PermountTM mounting medium (Thermo Fisher Scientific) and observed using an Olympus IX73 inverted microscope system.
6
Quantifying COX Isoforms in Cultured Cells
7
Sporoderm Breakage Analysis of Germinated Spores
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Powder of each sample (2.0 mg) was soaked in 1.0 mL of 30% ethanol. Before observing, each mixture was fully oscillated, taken 10 μL to observed under 40-fold objective lens (Olympus IX73 Inverted Microscope System, Tokyo, Japan). Each mixture was observed three times and averaged, samples from a same origin were compared, sporoderm-broken rate of broken GSP samples was calculated by following equation: (N1 = the average of spore from unbroken GSP sample; N2 = the average of unbroken spore from broken GSP sample).
8
Amyloid Fibril Formation Analysis of FUS RRM
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The samples of FUS RRM for assessing the formation of amyloid fibrils were prepared as previously reported14 (link), except for additional addition of ATP at different concentrations. The incubation samples were checked every day by differential interference contrast (DIC) and imaged at different time points by EM14 (link). Briefly, aliquot of 40 µL of incubation samples was used for DIC check every day by a DIC microscopy (OLYMPUS IX73 Inverted Microscope System with OLYMPUS DP74 Color Camera) or imaged by a TEM microscopy (Jeol Jem 2010f Hrtem, Japan) operating at an accelerating voltage of 200 kV as we previously described14 (link),41 (link).
9
Immunofluorescence Analysis of Angiogenic Markers
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Cells pooled from 6 wells per group were plated at the same time onto sterile 16-well culture slides (Fisher Scientific, Pittsburgh, PA, USA) and exposed to similar conditions as described above for the 24-well plates. At the end of each experimental time, 24 h, 48 h, and 72 h, the slides were washed, fixed in 4% paraformaldehyde, permeabilized, and incubated with HIF1α (rabbit polyclonal IgG, 1:200, VEGF (rabbit polyclonal IgG, 1:200), VEGFR-1 (goat polyclonal IgG, 1:200), VEGFR-2 (rabbit polyclonal IgG, 1:200), VEGFR-3 (rabbit polyclonal IgG, 1:200), NP (mouse monoclonal IgG, 1:200), Notch-1 (rabbit polyclonal IgG, 1:200), Notch-4 (rabbit polyclonal IgG, 1:200), DLL-4 (rabbit polyclonal IgG, 1:200), and Jagged-1 (rabbit monoclonal IgG, 1:200 primary antibodies purchased from Invitrogen Thermo Fisher (Waltham, MA, USA), Antibodies Online (Limerick, PA, USA), MyBioSource (San Diego, CA, USA), Novus Biologicals (Centennial, CO, USA) and Santa Cruz Biotechnology (Dallas, TX, USA)). Alexa Fluor fluorescent secondary antibodies (Life Technologies, Grand Island, NY, USA). Cells were imaged at 20× magnification using an Olympus IX73 inverted microscope system and CellSens imaging software (Olympus, Center Valley, PA, USA).
10
TUNEL Assay for Apoptosis in Piglets
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The TUNEL method was performed to detect the apoptotic changes in the jejunum of piglets as described by Duan et al.60 (link). Briefly, paraffin sections were deparaffinized in xylene, dehydrated through graded alcohols, and washed in freshly prepared deionized water. Then, the deparaffinized sections were treated with proteinase K for 30 min at 37 ◦C and rinsed in PBS. A methanol solution containing 3% hydrogen peroxide was used to block endogenous peroxidase for 5 min. Sections were immersed in a buffer containing terminal deoxynucleotidyl transferase and digoxigenin-labelled nucleotides for 2 h at 37◦C. After washing with PBS, sections were incubated with a pre-diluted anti-digoxigenin peroxidase-conjugated antibody for 30 min. Apoptotic cells were detected after the sections were incubated in the 3,3′-diaminobenzidine (DAB) chromogen for approximately 6 min and counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Imaging was performed by using the Olympus IX73 inverted microscope system.
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