The largest database of trusted experimental protocols

4 protocols using cell m imaging software

1

Dendritic Arbor Imaging via Epifluorescence and Spinning Disc Confocal Microscopy

Check if the same lab product or an alternative is used in the 5 most similar protocols
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).
+ Open protocol
+ Expand
2

Immunofluorescence Analysis of Neuronal Markers

Check if the same lab product or an alternative is used in the 5 most similar protocols
Immunofluorescence analysis was performed as previously described (Posada-Duque et al. 2013 (link)). Briefly, the cells were incubated overnight at 4°C with the following primary antibodies: mouse PSD95 (1:250, Calbiochem), rabbit PSD95 (1:250, Cell Signaling) and mouse MAP2 (1:2000, Sigma). The Alexa-594 fluorescent antibody was used as a secondary antibody (1:1000, Molecular Probes). The nuclei were stained with Hoechst 33258 (1:5000, Invitrogen), and the cells were incubated with phalloidin conjugated with Alexa 594 (1:2000, Molecular probes) for 1 h. The cells were washed 4 times in buffer, coverslipped using Gel Mount (Biomeda), and observed under an Olympus IX 81 epifluorescence microscope or a DSU Spinning Disc Confocal microscope. No staining was observed when the primary antibodies were omitted.
XY images were collected using an Olympus IX 81 microscope with 10× (NA, 0.4), 40× (NA, 1.3) or 60× (NA, 1.42) oil-immersion objectives. Z-stack images were collected at 0.4-µm intervals on an Olympus IX 81 DSU Spinning Disc Confocal microscope (60× oil-immersion; NA, 1.42). The image stacks were deconvolved using CellM imaging software (Olympus).
+ Open protocol
+ Expand
3

Mitotic Chromosome Enrichment and Imaging

Check if the same lab product or an alternative is used in the 5 most similar protocols
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.
+ Open protocol
+ Expand
4

Immunofluorescence Imaging of PAD Enzymes

Check if the same lab product or an alternative is used in the 5 most similar protocols
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 CellM imaging software (Olympus Corporation) provided with the Olympus 1X81 fluorescence microscope (Olympus Corporation).
+ Open protocol
+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required

Sign up now

Revolutionizing how scientists
search and build protocols!