Orca er cooled camera

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).

Immunostaining of fibroblast cells was accomplished as described previously^{103 (link),104 (link)} using the mouse anti-SMN monoclonal antibody (mAb) (MANSMA2 (8F7); Developmental Studies Hybridoma Bank, Iowa City, IA^{105 (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.

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.