Staining to detect senescence-associated beta-galactosidase (SA-βgal) activity was carried out as described previously (Debacq-Chainiaux et al., 2009 (link)). Briefly, cells were plated on 12-well plates, treated as indicated, subsequently fixed in 4% paraformaldehyde for 10 min, then incubated at 37°C for 12 to 24 hr in staining solution containing 1 mg/ml X-gal (Sigma-Aldrich). Images were taken on a Leica DMI600 microscope and analysed using CellProfiler software (Carpenter et al., 2006 (link)).
Dmi600 microscope
Manufactured by Leica
Sourced in United States, Switzerland
The Leica DMI600 is a digital microscope designed for laboratory applications. It features a high-quality optical system and advanced imaging capabilities. The DMI600 is capable of various imaging techniques, including brightfield, darkfield, phase contrast, and fluorescence microscopy.
Automatically generated - may contain errors
Lab products found in correlation
Las x software, by Leica (3 mentions)
Dfc345 fx camera, by Leica (2 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (2 mentions)
Dfc350fx r2 digital camera, by Leica (2 mentions)
X gal, by Merck Group (1 mentions)
Dmi6000, by Leica (1 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
Tcs spe confocal microscope, by Leica (1 mentions)
Alexa 555, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 anti rabbit, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 anti mouse, by Thermo Fisher Scientific (1 mentions)
Sab4200181, by Merck Group (1 mentions)
Rabbit anti ha tag 3724, by Cell Signaling Technology (1 mentions)
9 protocols using dmi600 microscope
1
Senescence-Associated Beta-Galactosidase Assay
2
Wound Healing Assay Protocol
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Cells were cultured in 24-well TCPS at the density of 20,000/cm2 for five days to reach confluence. The well surface was gently scratched with a pipette tip to create a rectangular scratch. After removal of culture medium and detached cells, remaining cells were washed twice with PBS and cultured in DMEM with 10% FBS. The cell images were photographed under Leica DMI600 microscope, and closure of the wound was analyzed using ImageJ software.
3
Quantifying Cellular Protein Localization
4
Leica DMI600 Microscope Imaging Protocol
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For light microscopy, images were obtained using a Leica DMI600 microscope and Leica LAS-x software and processed as previously described [11 (link)].
5
Indirect Immunofluorescence Microscopy of Peroxisomes
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Cells were prepared for indirect IF as previously described,29 (link) mounted onto slides using ProLong Gold Antifade Reagent with DAPI (Invitrogen, Waltham, MA, USA), and visualized using a Leica DMI600 microscope with a DFC345FX camera and LASX software (Leica, Richmond Hill, ON, Canada). Primary antibodies used were 1:300 rabbit anti-PTS1 (generated and gifted by Dr. Steven Gould, Johns Hopkins University, Baltimore, MD, USA), 1:150 mouse anti-human ABCD3 (SAB4200181; Sigma-Aldrich, St. Louis, MO, USA), and 1:300 rabbit anti-HsPEX5 (generated and gifted by Dr. Gabriele Dodt, University of Tübingen, Germany). Secondary antibodies used were 1:400 Alexa Fluor 488 anti-rabbit (A21206; Invitrogen, Waltham, MA, USA) and 1:300 Alexa Fluor 594 anti-mouse (A11005; Invitrogen, Waltham, MA, USA).
6
HA-Tagged Protein Immunohistochemistry in Mouse Retina
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For cryosections, eye cups from PBS-perfused mice were fixed 3 h in 10% formaldehyde; incubated in 10% (30 min on ice), 20% (1 h on ice), and 30% (4°C overnight) sucrose in 0.1 M PB; and then embedded and frozen in frozen-section compound (VWR, Mississauga, ON, Canada). 5 μm retinal cryo-sections were blocked (1% normal goat serum, 0.1% Triton X-100, 10% bovine serum albumin [BSA] in PBS) for 1 h, washed, incubated at 4°C overnight with primary antibody in incubation buffer (0.1% Triton X-100, 10% BSA in PBS), washed, incubated 90 min with secondary antibody, and washed. Coverslips were mounted using ProLong Gold antifade reagent with DAPI (Invitrogen, Burlington, CA, USA), and retinas were visualized using a Leica DMI600 microscope with a DFC345FX camera and LASX software (Leica, Richmond Hill, ON, Canada). The primary antibody used was 1:300 rabbit anti-HA tag (3724; Cell Signaling Technology, Danvers, MA, USA).
7
Quantifying Cell Surface Glycans in Murine Lung Endothelial Cells
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Mouse lung endothelial cells were plated on gelatin-coated coverslips and grown to confluence. On the day of an experiment, cells were washed with HBSS and fixed in 4%PFA for 20 min. Cells were blocked with 5% normal goat serum in PBS-T and incubated with primary antibodies (at 1:100 dilution for syndecan 1, syndecan 4, and heparan sulfate) in a wet chamber overnight at 4 °C. Negative controls were performed by incubating cells with blocking buffer overnight at 4 °C. The next day, cells were washed, blocked for 1 h with blocking buffer (5% normal goat serum in PBS-T), and then incubated with the secondary antibody (1:500 dilution in PBST) overnight at 4 °C. On the following day, cells were rinsed with PBS-T, incubated with DAPI 1µg/mL for 15 min at room temperature, washed three times for ten minutes with PBS-T, and mounted in in Prolong antifade mounting medium. Images were taken using the LEICA DMI600 microscope at The University of Arizona Imaging Core using LAS X software V. 3.7 (Leica, IL, USA); 2D, 2.5D, and 3D views were constructed using LAS X software V.1.4.5 (Leica, IL, USA). Equal brightness and contrast were applied to all images. The density of heparan sulfates, syndecan 1, and syndecan 4 in MLECs was assessed by measuring fluorescence/area using Image J software V.1.53e (developed at the U.S. National Institutes of Health), and data are shown as mean ± SEM.
8
Quantifying Placental SEAP and EYFP
9
Quantifying Placental SEAP and EYFP
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The production of human placental SEAP was quantified in cell culture supernatants39 (link) and mouse serum40 (link) as described previously. EYFP expression was visualised using a LEICA DMI-600 microscope (Leica Microsystems, Heerbrugg, Switzerland) equipped with a DFC350FX R2 digital camera (Leica), a 10x objective, a 488 nm/509 nm (B/G/R) excitation/emission filter set and Leica Application Suite software (version V2.1.0R1).
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