Matrigel plugs or muscles were collected, fixed with 4% paraformaldehyde, and embedded in OCT. 8 µm (muscle) and 10 µm (Matrigel plugs) sections were cut using a cryostat and immediately transferred to poly‐L‐lysine coated slides. Permeabilization buffer containing 1% BSA and 0.4% Triton X‐100 in PBS was used to permeabilize tissue. Non‐specific binding was blocked by incubating the tissue sections with 3% BSA in PBS‐T for 30 min at room temperature. Tissue sections were then incubated with a goat anti‐mouse CD31 antibody (R&D, #AF3628) overnight in a humidified chamber, followed by Donkey anti‐goat green IgG NorthernLights NL493‐conjugated secondary Antibody (R&D system, #NL‐493). Sections were mounted with ProLong Gold Antifade Mountant with DAPI (Invitrogen). Fluorescence images were recorded on three random fields per section with an EVOS FL auto inverted microscope (Life Technologies).
Evos fl auto inverted microscope
Manufactured by Thermo Fisher Scientific
The EVOS FL auto inverted microscope is a compact, automated fluorescence imaging system designed for a wide range of live-cell and fixed-sample applications. It features LED illumination, automated image capture, and onboard image analysis capabilities.
Automatically generated - may contain errors
Lab products found in correlation
Prolong gold antifade mountant with dapi, by Thermo Fisher Scientific (2 mentions)
Af3628, by R&D Systems (1 mentions)
Avidin biotin blocking kit, by Vector Laboratories (1 mentions)
Evos software, by Thermo Fisher Scientific (1 mentions)
Citrisolv, by Thermo Fisher Scientific (1 mentions)
Reveal decloaker, by Biocare Medical (1 mentions)
Sk 4100, by Vector Laboratories (1 mentions)
Vectastain elite abc kit, by Vector Laboratories (1 mentions)
Dm6000 microscope, by Leica (1 mentions)
Fv1000 microscope, by Olympus (1 mentions)
5 protocols using evos fl auto inverted microscope
1
Immunofluorescent Analysis of Angiogenesis
2
HMGB1 Immunohistochemistry in Mouse Ovary
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Ovaries from 40-day-old CD1 mice were fixed in Modified Davidson's Fixative (Electron Microscopy Sciences, 64133-50) and paraffin-embedded. Five micron sections were deparaffinized in Citrisolv (Fisher, 04-355-121) and antigen retrieval achieved using Reveal Decloaker (Biocare Medical, RV1000). Sections were prepared for immunohistochemistry using Avidin/Biotin blocking kit (Vector Labs, SP-2001) in 10% normal goat serum in TBS. Sections were then incubated with diluted 1:100 HMGB1 primary antibody (Abcam, ab18256, lot GR135551-1) in 10% normal goat serum in TBS overnight at 4 °C. As a control for expression, the HMGB1 blocking peptide (Abcam, ab18650) was also used. A ratio of 1:5 HMGB1 antibody to HMGB1 peptide was incubated overnight at 4 °C prior to application. Sections were washed in TBS+0.1% Tween-20 and then incubated with diluted 1:200 biotinylated goat anti-rabbit secondary antibody from the Vectastain Elite ABC kit (Vector Labs, PK-6105) for 2 h at room temperature, washed again and followed by a 30 min incubation in the ABC reagent (Vector Labs, Vectastain Elite ABC kit) and a final wash. Binding was detected with diaminobenzidine (DAB; Vector Labs, SK-4100) for 6 min. Counterstaining was achieved using standard haematoxylin staining. All images were acquired and processed on an Evos fl auto inverted microscope using Evos software (Life Technologies).
3
Immunohistochemical Analysis of Angiogenesis
4
Cell Morphology Analysis Protocol
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Cell images were captured at different passages at 48 h post seeding using EVOS-FL auto inverted microscope (Life Technologies) at 10× magnification. Cell spreading area was determined using ImageJ (National Institutes of Health) software by manually tracing around the perimeter of an individual cell. For each sample minimum, 150 random cells were analyzed. The number of protrusions of cells was quantified from phase contrast images manually using ImageJ.
5
Multimodal Imaging of Cell Clones
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Epifluorescence images were collected with a 10 × 0.6 NA or 20 × 0.7 NA objective on a Leica DM6000 microscope equipped with a VT1000 camera and separate filter cubes for GFP, RFP and IRFP. Confocal image stacks were acquired with 20 × 0.8 NA oil and 40 × 1.25 NA silicone objectives on an Olympus FV1000 microscope, using 440, 488, 515, 560, and 633 nm laser lines to excite CFP, GFP, YFP, RFP and IRFP/Alexa 647, respectively. For analysis with Arrayscan (Thermo Fisher Scientific), cells grown in 24- or 96-well plates were fixed 15 min with 4% PFA and stained with 300 nM DAPI prior imaging with the following laser lines: 386 nm (DAPI), 485 nm (GFP), 570 nm (RFP), and 650 nm (IRFP). Images of live ES cell clones were acquired using an EVOS FL auto inverted microscope (Life Technologies). Image analysis was performed with Fiji (Schindelin et al., 2012 (link)) and Imaris (Bitplane). Levels were uniformly adjusted across images with Adobe Photoshop.
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