Tissue Imaging and Colocalization Protocol

Conducted as described previously^{16 (link)}. Briefly, tissues for imaging were fixed overnight in (mouse) or for 7 days (NHP) in 10% neutral buffered formalin (Sigma, HT501320), and then transferred to PBS. All tissues were paraffin imbedded and sliced into 4 μM sections. Sections were stained with DAPI and imaged on a Leica inverted microscope. Staining for colocalization of Di-siRNA with neuronal (NueN) and glial cells (GFAP) was performed as described previously^{50 (link)}.

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Alterman J.F., Godinho B.M., Hassler M.R., Ferguson C.M., Echeverria D., Sapp E., Haraszti R.A., Coles A.H., Conroy F., Miller R., Roux L., Yan P., Knox E.G., Turanov A.A., King R.M., Gernoux G., Mueller C., Gray-Edwards H.L., Moser R.P., Bishop N.C., Jaber S.M., Gounis M.J., Sena-Esteves M., Pai A.A., DiFiglia M., Aronin N, & Khvorova A. (2019). A divalent siRNA chemical scaffold for potent and sustained modulation of gene expression throughout the central nervous system. Nature biotechnology, 37(8), 884-894.

Publication 2019

HT501320 inverted microscope

Dapi Formalin Gfap Glial cells Microscope Mouse Neuronal Paraffin Sirna Tissues

Corresponding Organization : University of Massachusetts Chan Medical School

Other organizations : Institute for Neurodegenerative Disorders

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Variable analysis

independent variables

Tissue type (mouse vs NHP)

dependent variables

Cellular localization of Di-siRNA

control variables

Tissue fixation protocol (10% neutral buffered formalin, overnight for mouse and 7 days for NHP)
Tissue sectioning (4 μM thickness)
Staining (DAPI)
Imaging (Leica inverted microscope)

controls

Positive control: Staining for neuronal (NueN) and glial cells (GFAP) to colocalize with Di-siRNA, as described previously in reference 50.
Negative control: Not explicitly mentioned.

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