Immu mount aqueous mounting medium

Following in vitro culture or in vivo implantations, samples were fixed in a solution of 4% paraformaldehyde overnight. Subsequent to fixation, some samples were decalcified in a solution of 0.4 M ethylenediaminetetraacetic acid (EDTA) for 1–2 h. Samples were embedded in paraffin, and 5 μm longitudinal sections were prepared. Sections were stained for hematoxylin and eosin (H&E) to characterize cell and tissue morphology and structure, and un-decalcified sections were also stained for Alizarin Red to detect mineralization. Immunostaining was performed to detect bone sialoprotein (BSP; Millipore); periostin (Novus); periodontal ligament-associated protein 1 (PLAP-1), also known as asporin (Invitrogen); or alkaline phosphatase (ALP; Abcam). Fluorescently tagged secondary antibodies, Alexa Fluor 546 goat anti-mouse and Alexa Fluor 488 goat anti-rabbit (Invitrogen), were used to detect the signal. Negative controls to our immunostaining were performed in parallel by omitting the primary antibody. DAPI (4′,6-diamidino-2-phenylindole, dihydrochloride) staining was performed to detect nuclei, and slides were mounted with Immu-mount aqueous mounting medium (Thermo Scientific). Brightfield images were acquired using a Nikon Eclipse TE200-E inverted microscope, and fluorescent images were taken using a Nikon Eclipse Ti inverted microscope.

Cells were plated over sterilized coverslips in 6-well plates and transfected as above using mCherry-FUS, pEGFP-LMNA and myc- or FLAG-tagged SETX constructs. Constructs were expressed for 72 h in culture medium as described above. Coverslips were then washed with 1 × PBS and fixed for 15 min in 4% formaldehyde in PBS at RT, washed gently 3 times with 1 × PBS, and permeabilized with 2% Triton-X-100 in 1 × PBS for 10 min. Blocking was performed with 10% bovine serum albumin Fraction V (ThermoFisher) for 1 h at room temperature. Primary antibodies (as needed to detect SETX and endogenous proteins; see Supplemental Table 1 for all antibodies used in these studies) were then added and incubated for 1 h at RT. Cells were washed in PBS twice for 10 min each and then incubated with secondary antibody (see Supplemental Table 1) for 1 h. Cells were washed again in PBS then incubated with Hoechst for 5 min, washed twice in PBS for 5 min. For cells that did not require antibodies for detection of expressed proteins from transfected constructs, cells were fixed, washed in PBS, stained with Hoechst without permeabilization or incubation steps. Coverslips were then inverted and mounted to glass slides using Immu-Mount aqueous mounting medium (ThermoFisher) and imaged using a SP8 Leica confocal microscope.