Insulin and glucagon protein abundance was assessed using paraffin-embedded pancreatic sections from WT, PdxCreMpc2−/−, and RipCreMpc2−/− mice. Slides were rehydrated, permeabilized with 1 mg/ml trypsin, blocked in 3% BSA, and probed with guinea pig anti-insulin (Abcam #ab10988, 1:100 dilution) and mouse anti-glucagon (Abcam #ab7842, 1:100 dilution) antibodies, in 3% BSA overnight. Slides were washed and probed with Alexa Fluor® 488 goat anti-guinea pig (Invitrogen #A11073, 1:1000 dilution) and Alexa Fluor® 594 goat anti-mouse secondary (Invitrogen #A21125, 1:1000 dilution) antibodies in 3% BSA for 1 h. Coverslips were fixed with ProLong® Gold antifade reagent with DAPI (Life Technologies). Stained pancreatic sections were then imaged on an EVOS FL digital inverted fluorescence microscope (Invitrogen). Islet number and cross-sectional areas were determined using ImageJ.
Evos fl digital inverted fluorescence microscope
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
The EVOS™ FL Digital Inverted Fluorescence Microscope is a high-performance digital microscope designed for fluorescence imaging. It features a compact, inverted design and a digital camera for capturing and recording images and videos of fluorescently labeled samples.
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Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (6 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Methocult express, by STEMCELL (2 mentions)
Anti hfabp4, by R&D Systems (2 mentions)
Anti hoc, by R&D Systems (2 mentions)
Anti hacan, by R&D Systems (2 mentions)
Alexa flour 488 goat anti mouse, by Thermo Fisher Scientific (2 mentions)
Chondrogenic supplement, by R&D Systems (2 mentions)
Ab7842, by Abcam (1 mentions)
Ab10988, by Abcam (1 mentions)
Alexa fluor 488 goat anti guinea pig, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
Pezgs0416, by Merck Group (1 mentions)
Evos light cube, by Thermo Fisher Scientific (1 mentions)
Prolong diamond antifade mountant with dapi, by Thermo Fisher Scientific (1 mentions)
Ferroorange, by Dojindo Laboratories (1 mentions)
Spectramax i3x multi mode microplate reader, by Molecular Devices (1 mentions)
Hoechst dye, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Dmem f12, by R&D Systems (1 mentions)
29 protocols using evos fl digital inverted fluorescence microscope
1
Quantification of Pancreatic Insulin and Glucagon
2
3D Coculture Spheroid Formation Assay
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3D coculture spheroid formation assay was performed as previously described23 (link). Briefly, mouse F4/80+CD11b+ TAMs isolated from OC-bearing mice and ID8 tumor cells were mixed at a ratio of 1:10 with a fixed total number of 40,000 cells/well in media containing 2% Matrigel and seeded onto Matrigel-precoated 24-well plate. The cell mixtures were incubated at 37 °C for 48 h and the number (per well) and size (area) of spheroids were quantified. Images were captured with Invitrogen™ EVOS™ FL Digital Inverted Fluorescence Microscope and analyzed using ImageJ software.
3
Histological Analysis of Brown and Subcutaneous Adipose Tissue
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After necropsy, brown adipose tissue and subcutaneous (SubQ) fat were harvested from the mice and immediately fixed in 10% buffered formalin. The fixed tissues were then embedded in paraffin, and 5–7 μm sections were prepared for H&E staining using established methods described in a previous study by60 (link). Bright-field images of the stained sections were captured using an Invitrogen microscope (Invitrogen™ EVOS™ FL Digital Inverted Fluorescence Microscope; Invitrogen, Thermo Fisher Scientific) at magnifications of 10× and 20×.
4
Neural Crest Cell Migration Assay
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The embryos were isolated on the embryonic day 9.0 (E9.0). The vagal neural tubes (somites 1–7), and the trunk neural tubes (somites 9–12), were dissected out by removing the surrounding tissues by using micro-scissors. The neural tube explants were then placed on fibronectin coated cover slips in N2 supplemented DMEM media (both from Gibco, Thermo Fisher Scientific). The explants were incubated at 37 °C with 5% CO2 for 48 h during which the NC cells migrated out of the NT explant and formed a halo. The migration length was measured as the furthest distance the NC cells had reached away from the explant at 48 h. The measurements of each halo consisted of twenty-five data points evenly covering the entire halo of NC cells (Fig. 5 A–D). The images were acquired at 48h using the EVOS™ FL digital inverted fluorescence microscope (Invitrogen). The images were analyzed by using the ImageJ software (http://rsb.info.nih.gov/ij/ , 1997–2009; Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, MD, USA).
5
Quantifying Intracellular Iron Levels
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Cells (2 × 104) were plated on 4-well chamber slides (PEZGS0416; Millipore) coated with 2 µg/mL fibronectin (CB4008; BD Biosciences). The cells were incubated with 10 µM DFO (Sigma) or equal volume of PBS as a vehicle control for 48 h. Then, the cells were washed with serum-free DMEM three times and incubated with 1 µM FerroOrange (F374-10; Dojindo, Rockville, MD) prepared in serum-free EC culture medium for 30 min in a 37 °C incubator. The cells were photographed with an inverted fluorescence microscope (EVOS FL Digital Inverted Fluorescence microscope; Invitrogen). Mean fluorescence intensities were measured from 5 images per group.
6
Nanoemulsion Uptake in Mouse Macrophages
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Images of mouse macrophages labeled with nanoemulsion were acquired using the EVOS™ FL Digital Inverted Fluorescence Microscope from Invitrogen. RAW 264.7 macrophages were cultured on glass chamber slides (Lab Tek Chamber Slides, 8 well slides) at 50,000 cells/well and incubated for 24 h. Macrophages were treated with nanoemulsion containing 25 µM DiR and 10 µM DiI at a dilution of 10 µL nanoemulsion in 1 mL of culture medium and were incubated for 24 h at 37 °C and 5% CO2. After removing the treatment media, macrophages were fixed with 0.3 mL 4% paraformaldehyde (PFA) in PBS for 10 min. The PFA was then removed and the macrophages were washed 3 times with PBS. The chambers were removed and a cover slip was mounted onto the slide using a mountant containing DAPI (ProLong™ Diamond Antifade Mountant with DAPI, Molecular Probes). Images were captured using the EVOS Microscope using the white light, DAPI, and Red Fluorescent channels. For visualizing, the DAPI channel, Invitrogen™ EVOS™ Light Cube, DAPI was used (excitation of 357/44 and emission of 447/60). For visualizing nanoemulsion uptake into the macrophages, Invitrogen™ EVOS™ Light Cube, RFP was used (excitation 531/40 and emission 593/40). Images of mouse macrophages treated with nanoemulsion can be seen in Fig. 3 A–C.
7
Quantitative Assessment of Cellular Iron Levels
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A total of 2.0 × 104 cells were plated on fibronectin-coated 4-well chamber slides (2 µg/mL in serum-free DMEM). The next day, the cells were rinsed with serum-free medium three times and incubated with 1 µM FerroOrange (F374-10; Dojindo, Rockville, MD, USA) for 30 min in a 37 °C incubator. The cells were photographed with an inverted fluorescence microscope (EVOS FL Digital Inverted Fluorescence microscope (Invitrogen). For quantitative assessment of the data, the mean integrated fluorescence intensities were determined from at least 6 images from different locations of the slides using ImageJ. Alternatively, 3.0 × 104 cells were plated on 96-well plates and stained with FerroOrange as described above. The fluorescence levels were measured by a fluorescence plate reader (SpectraMax i3x Multi-mode Microplate Reader, Molecular devices) at 543/580 nm and normalized to total protein levels determined by BCA protein assay (ThermoFisher).
8
Adipose Tissue Analysis and DPPH Assay
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Upon necropsy, epididymal adipose tissue was collected from the mice and flash-fixed in 10% buffered formalin. The paraffin-embedded adipose tissues were cut into 5-7 μm sections and processed for hematoxylin and eosin (H&E) staining, as described previously. 27 Bright-field images were obtained by an Invitrogen microscope (Invitrogen™ EVOS™ FL Digital Inverted Fluorescence Microscope, Invitrogen, CA, USA) under 10× magnification. H&E-stained sections of epididymal adipose tissue were quantified for measuring adipocyte size. Image J and Adiposoft from the National Institutes of Health (NIH) were used for the adipocyte size quantification.
2,2-Diphenyl-1-picryl-hydrazyl-hydrate (DPPH) of PSE and PCA
The DPPH radical scavenging activity was measured by the previously described method with some modifications. 28, 29 (link) Briefly, DPPH was dissolved in ethanol to a 0.2 mM concentration. In a 96-well plate, 25 μL of the samples (6 mg mL -1 of PSE or PCA), and 175 μL of DPPH solution were incubated at 37 °C for 30 min. The absorbance of the samples was measured at 517 nm. Ascorbic acid (6 mg mL -1 ) was used as a positive control. The free radical scavenging activity (DPPH) was calculated according to the following formula:
Free radical scavenging activityð%Þ ¼ ½1 À ðabsorbance of sample =absorbance of controlÞ Â 100:
2,2-Diphenyl-1-picryl-hydrazyl-hydrate (DPPH) of PSE and PCA
The DPPH radical scavenging activity was measured by the previously described method with some modifications. 28, 29 (link) Briefly, DPPH was dissolved in ethanol to a 0.2 mM concentration. In a 96-well plate, 25 μL of the samples (6 mg mL -1 of PSE or PCA), and 175 μL of DPPH solution were incubated at 37 °C for 30 min. The absorbance of the samples was measured at 517 nm. Ascorbic acid (6 mg mL -1 ) was used as a positive control. The free radical scavenging activity (DPPH) was calculated according to the following formula:
Free radical scavenging activityð%Þ ¼ ½1 À ðabsorbance of sample =absorbance of controlÞ Â 100:
9
DCVC Impacts Mitochondrial Membrane Potential
10
Optimizing K562 Cell Colony Formation
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According to the manufacturer’s instructions, colony formation experiments were conducted using MethoCult® Express (Catalog #04437; STEMCELL Technologies, Vancouver, BC, Canada) methylcellulose-based culture medium according to the manufacturer’s instructions. Briefly, 1 × 104 K562 or K562 PR cells were plated on MethoCult™ Express with the indicated concentrations of asciminib and/or VK2. The plates were incubated at 37 °C and 5% CO2 for 7 days. Colony counts were calculated, and imaging was conducted with an EVOS™ FL Digital Inverted Fluorescence Microscope (Thermo Fisher Scientific Inc., Waltham, MA, USA). The results were determined three times, and the mean and standard error were displayed.
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