Immunostaining of Fibroblast Cells for SMN

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.

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Lumpkin C.J., Harris A.W., Connell A.J., Kirk R.W., Whiting J.A., Saieva L., Pellizzoni L., Burghes A.H, & Butchbach M.E. (2023). Evaluation of the orally bioavailable 4-phenylbutyrate-tethered trichostatin A analogue AR42 in models of spinal muscular atrophy. Scientific Reports, 13, 10374.

Publication 2023

DMRXA2 epifluorescence microscope ORCA-ER cooled camera Volocity 6.1.1 software

Anti antibody Buffalo Fibroblasts Gems Hybridoma Microscope Monoclonal antibody Mouse Nuclei Orca

Corresponding Organization :

Other organizations : Dupont Hospital, DuPont (United States), Columbia University, The Ohio State University Wexner Medical Center, University of Delaware

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Variable analysis

independent variables

Treatment of fibroblast cells

dependent variables

SMN immunostaining within the nuclei of treated fibroblasts
Number of gems
Number of cells with gems
Number of cells with more than 1 gem

control variables

Immunostaining protocol as described previously in references 103 and 104
Mouse anti-SMN monoclonal antibody (mAb) (MANSMA2 (8F7)) used for immunostaining
Visualization of SMN immunostaining using DMRXA2 epifluorescence microscope, ORCA-ER cooled camera, and Volocity 6.1.1 software

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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