Gelatin degradation assays were imaged using a LEICA- DMRXA2 epifluorescence microscope with a 40× PL Fluotar (O.N.; 1.00) oil objective. Confocal imaging used a LEICA SP8 DMI 6000 CS microscope using oil immersion objectives (40×/1.30 HC PL APO) and (63×/1.4 HC PL APO). Images were acquired using the LAS-AF (Leica, Wetzlar, Germany) software. Z-stacks were acquired with Surplatine superZ galvo at a step size of 500 nm. Brightness and contrast adjustments of images were realized using the imageJ software.
Dmrxa2 epifluorescence microscope
Manufactured by Leica
Sourced in Japan
The DMRXA2 is an epifluorescence microscope manufactured by Leica. It is designed for fluorescence imaging and analysis. The DMRXA2 features a modular design and supports a range of objectives and fluorescence filter sets to enable versatile imaging capabilities.
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Lab products found in correlation
5 protocols using dmrxa2 epifluorescence microscope
1
Gelatin Degradation Imaging Protocol
2
DAPI Staining of Chromosome Spreads
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Chromosome spreads were prepared and stained with DAPI as described in Ross et al. (1996) (link). Observations were made with a Leica DM RXA2 epifluorescence microscope using an oil PL APO 100X/1.40 objective (Leica). Chromosomes were counted on cells at mitotic metaphase.
3
Quantifying SMN Gems in Fibroblasts
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SMN immunostaining in fibroblasts was visualized using a DMRXA2 epifluorescence microscope (Leica Microsystems) with an ORCA-ER cooled camera (Hamamatsu, Hamamatsu City, Japan) and Volocity 6.1.1 software (Perkin-Elmer). For gem counting, the following parameters were measured in 100 randomly selected nuclei: the number of gems, the number of cells with gems and the number of cells with more than 1 gem. The gem counts were converted into a concentration value (in mM) using an approximate average cell volume (2.68×10−13 L). This conversion assumes that all cells are of equal volume [29] (link).
4
Immunostaining of Fibroblast Cells for SMN
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Immunostaining of fibroblast cells was accomplished as described previously103 (link),104 (link) using the mouse anti-SMN monoclonal antibody (mAb) (MANSMA2 (8F7); Developmental Studies Hybridoma Bank, Iowa City, IA105 (link)). SMN immunostaining within the nuclei of treated fibroblasts was visualized using a DMRXA2 epifluorescence microscope (Leica Microsystems Inc., Buffalo Grove, IL) with an ORCA-ER cooled camera (Hamamatsu, Hamamatsu City, Japan) and Volocity 6.1.1 software (Perkin-Elmer, Waltham, MA). For gem counting, the following parameters were measured in 100 randomly selected nuclei: the number of gems, the number of cells with gems and the number of cells with more than 1 gem.
5
Immunofluorescence Quantification of SMN Gems
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For immunofluorescence, cells were seeded onto gelatinized glass coverslips at a density of 4000 cells/cm2 and treated with compounds as described above. Immunostaining of fibroblast cells was accomplished as described previously [19 (link);35 (link)] using the MANSMA2 mouse anti-SMN mAb (1:200; Developmental Studies Hybridoma Bank, Iowa City, IA [36 (link)]). SMN immunostaining within the nuclei of treated fibroblasts was visualized using a DMRXA2 epifluorescence microscope (Leica Microsystems) with an ORCA-ER cooled camera (Hamamatsu, Hamamatsu City, Japan) and Volocity 6.1.1 software (Perkin-Elmer). Gems were counted 10 randomly selected nuclei in a field of view; this process was repeated for a total of 10 randomly selected, non-overlapping fields of view. The following parameters were measured: the number of gems, the number of cells with gems and the number of cells with more than 1 gem.
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