Images in the XY plane were collected using an Olympus IX 81 epifluorescence microscope equipped with a 60X (NA, 1.42) oil-immersion objective. Z-stack (6-10 optical sections) images were collected at 0.4-μm intervals encompassing the partial dendritic arbor using an Olympus IX 81 DSU Spinning Disc Confocal microscope equipped with a 60X oil-immersion objective (NA, 1.42). The image stacks were deconvoluted via a 3D-deconvolution algorithm following maximum intensity projection using Cell M imaging software (Olympus).
Cell m imaging software
Manufactured by Olympus
Cell M imaging software is a digital imaging application developed by Olympus. It is designed to capture, analyze, and manage digital microscope images. The software provides tools for image acquisition, processing, and data management.
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Lab products found in correlation
Ix 81 dsu spinning disc confocal microscope, by Olympus (2 mentions)
Ix81 epifluorescence microscope, by Olympus (2 mentions)
Alexa 594, by Thermo Fisher Scientific (1 mentions)
Dsu spinning disc confocal microscope, by Olympus (1 mentions)
Mouse map2, by Merck Group (1 mentions)
Hoechst 33258, by Thermo Fisher Scientific (1 mentions)
Ix81 microscope, by Olympus (1 mentions)
Karyomax colcemid, by Thermo Fisher Scientific (1 mentions)
Bx61 epifluorescence microscope, by Olympus (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Alexa fluor 488 conjugated anti rabbit igg secondary antibody, by Thermo Fisher Scientific (1 mentions)
Dapi vectashield medium, by Vector Laboratories (1 mentions)
4 protocols using cell m imaging software
1
Dendritic Arbor Imaging via Epifluorescence and Spinning Disc Confocal Microscopy
2
Immunofluorescence Analysis of Neuronal Markers
3
Mitotic Chromosome Enrichment and Imaging
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Forty-eight hours after transfection, subconfluent MDA-MB-231, MCF10A, and HEK cells were treated with KaryoMAX Colcemid (Invitrogen) to enrich for mitotic chromosomes. The complete medium was replaced by 2 ml of medium at a final concentration of KaryoMAX of 0.6 mg/ml. Cells were incubated at 37°C in 5% CO2 for 5 hours, harvested, trypsinized, pelleted (110g for 5 minutes), and resuspended in 1 ml of a hypotonic solution of 60 mM KCl and left at room temperature for 30 minutes. After incubation, the different cell lines were pelleted twice (110g for 5 minutes) and resuspended in freshly made methanol/glacial acetic acid (3:1) added dropwise. Two or three drops of suspended cells were applied to pre-cleaned smear glass slides (Menzel-Glazer, Braunschweig, DE) and chromosomes were counterstained with VECTASHIELD (Vector Laboratories) containing DAPI. Mitotic spreads of each cell population were imaged with the DAPI channel of a BX61 Olympus epifluorescence microscope equipped with a 1006/1.30 UPlan FLN objective coupled to a U-C MAD 3 imaging system with the Cell-M imaging software (Olympus). A minimum of 200 metaphases was evaluated per construct and metaphases in which > 70% of the chromosomes had separated sister chromatids were scored as positives.
4
Immunofluorescence Imaging of PAD Enzymes
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PC3 and PNT2 cells (5×105 cells/well) were seeded on a cover slip in a 24-well plate in triplicate, incubated for 24 h, washed gently with prewarmed PBS, fixed with 4% PFA for 10 min at RT, washed 3 times with cold PBS and resuspended in PB for 5 min at RT. The buffer was then removed and the cells were washed 3 times as before.
After incubation with PAD2 or PAD4 primary antibodies (1:500 dilution in 3% BSA/PBS) for 1 h at 4°C on a shaking platform, the cells were washed 3 times with cold PBS and further incubated with AlexaFluor 488 conjugated anti-rabbit IgG secondary antibody (Invitrogen; 5 µg/ml in 3% BSA/PBS) at 4°C for 1 h on a shaking platform in the dark. The cells were then washed 3 times with cold 1% BSA/PBS and the cover slips mounted on to slides with DAPI-VECTASHIELD medium (Vector Laboratories, Inc., Burlingame, CA, USA). Images were acquired using a fluorescence microscope (1X81 motorized inverted fluorescence microscope, Olympus Corporation, Hamburg, Germany). The mean green fluorescence intensity of the fluorescence images was analysed as per the manufacturer's instructions using the Cell∧M imaging software (Olympus Corporation) provided with the Olympus 1X81 fluorescence microscope (Olympus Corporation).
After incubation with PAD2 or PAD4 primary antibodies (1:500 dilution in 3% BSA/PBS) for 1 h at 4°C on a shaking platform, the cells were washed 3 times with cold PBS and further incubated with AlexaFluor 488 conjugated anti-rabbit IgG secondary antibody (Invitrogen; 5 µg/ml in 3% BSA/PBS) at 4°C for 1 h on a shaking platform in the dark. The cells were then washed 3 times with cold 1% BSA/PBS and the cover slips mounted on to slides with DAPI-VECTASHIELD medium (Vector Laboratories, Inc., Burlingame, CA, USA). Images were acquired using a fluorescence microscope (1X81 motorized inverted fluorescence microscope, Olympus Corporation, Hamburg, Germany). The mean green fluorescence intensity of the fluorescence images was analysed as per the manufacturer's instructions using the Cell∧M imaging software (Olympus Corporation) provided with the Olympus 1X81 fluorescence microscope (Olympus Corporation).
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