No 1.5 cover glass

Masters for the cell-bending device and the cell growth/osmotic shock device were fabricated on separate 3-in² silicon wafers using SU8 photoresist that was exposed on a Heidelberg μPG101 mask writer (Heidelberg Instruments, Heidelberg, Germany) and developed. The bending device (Fig. S1) used was a 2-layer device. The first SU8 layer consisted of ∼1-μm-tall channels for capturing cells. The wafer was then coated with the second SU8 layer (∼25 μm tall), which formed a central flow channel. The flow chamber (Fig. S12) consisted of a single channel with a length of 10 mm, a width of 5 mm, and a height of 50 μm. The volume of the flow chamber was ∼10 μl. After developing the masters for both devices, we used the masters to emboss layers of poly(dimethylsiloxane) (PDMS), punched holes for inlets and outlets, and cleaned the surfaces with tape. If the device was attached to a glass coverslip, it was treated with oxygen plasma and immediately sealed against a plasma-treated no. 1.5 cover glass (Fisherbrand) (24 by 50 mm) to form a permanent seal.

Immunohistochemistry methods have been described previously28 (link)29 (link)45 (link)67 (link). In brief, mice were fixed by transcardiac perfusion with 2% paraformaldehyde in PBS. Sternocleidomastoid muscle was dissected and post-fixed in the same fixative at room temperature, cryoprotected in 20% sucrose/PBS at 4˚C overnight, frozen in Optimal Cutting Temperature compound (Sakura), and sectioned using a cryostat. Longitudinal sections were cut at 20 μm thickness and blocked in PBS containing 2% BSA, 2% normal goat serum, and 0.1% Triton X-100. Sections were incubated with primary antibodies and biotin conjugated α-bungarotoxin overnight at room temperature, washed with PBS, and incubated with a mixture of secondary antibodies and Pacific Orange conjugated Streptavidin for 2 hours at room temperature. After washes with PBS, the sections were mounted in Prolong Gold mounting medium (Life Technologies, Carlsbad, CA, USA) with No. 1.5 cover glass (Fisher Scientific, Pittsburgh, PA USA). No staining was observed when primary antibodies were omitted.