Mouse anti smooth muscle actin

Stromal cells were isolated by FACS and plated onto 18-mm diameter circular coverslips in 12-well plates, and fixed 4% paraformaldehyde for 20 min. Cells were permeabilized with PBS/0.5% Triton X-100 for 3 min and nonspecific binding sites were blocked with 2% BSA in PBST (PBS with 0.5% Tween- 20) for 1 h. Cells were stained with primary antibodies: chicken anti-vimentin (Millipore AB5733, 1:600) and mouse anti-smooth muscle actin (Sigma 1A4, 1:1000) diluted in 2% BSA/PBST for 1 h at room temperature. After washing four times in PBST, cells were incubated for 1 h with appropriate Alexa fluor 488 or 594-conjugated secondary antibodies (Invitrogen, 1:1000) together with 1 g/mL of 4,6-diamidino-2-phenylindole (DAPI) for nuclear staining in 2% BSA/PBST. Cells were washed four times in PBST, then mounted on slides with Prolong Gold mounting reagent (Invitrogen). Immunofluorescence images presented are images from one representative experiment of three.

Frozen tissue sections were incubated in 1xPBS at room temperature to remove O.C.T, permeabilized with 0.3% vol/vol Triton-X100in 1xPBS. Primary and secondary antibodies were diluted in blocking buffer (1xPBS with: 5% vol/vol normal donkey serum, 0.3% vol/vol Triton-X100). Sections were stained with primary antibody at 4°C overnight (primary antibody dilutions: 1:500 Mouse anti-smooth muscle actin, Sigma-Cat# F1840–200uG; 1:500 Mouse antiHuC/D IgG2b, Invitrogen-Cat#A21271; 1:10 E1.9 primary sensory and motor neuron marker, DSHB). The next day, sections were washed with 1xPBS, and treated with secondary antibody for 1 hour at room temperature. The following secondary antibodies were used: 1:1000 donkey anti-mouse IgG 647, 1:1000 goat anti-mouse IgM 647, 1:1000 goat anti mouse IgG2b 647, Molecular Probes. Sections were imaged using a Zeiss AxioImager.M2 with Apotome.2 and Zeiss LSM 800 confocal microscope. The number of infected cells displaying single color was quantified with thresholding followed by automated particle analysis using FIJI software. The number of doubly and triply infected cells was counted manually. For Feret’s angle analysis of clonal orientation, a cell cluster with identical double-or triple- color was identified. Subsequently, a polygon was drawn along the boundary of the clone and the angle of the polygon was automatically measured by FIJI.