Embed 812 hard resin

Plasmids were transfected into HeLa cells growing on 100 mm dishes using X-treme GENE HP DNA transfection reagent (Roche, Laval, QC) according to manufacturer’s protocols and processed for TEM at 18 hours post transfection. Transfected cells were washed with PBS three times before fixing in 1.5 ml of 2.5% EM grade glutaraldehyde (Ted Pella, Redding, CA) in 0.1 M sodium cacodylate buffer (pH 7.4) for 60 minutes. Cells were collected by scraping into fixative and centrifugation at 300 x g for five minutes. Cell pellets were carefully enrobed in an equal volume of molten 5% low-melting temperature agarose (Lonza, Rockland, ME) and allowed to cool. Specimens in agarose were incubated in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 1.5 hours and post-fixed in 1% osmium tetroxide for one hour. The fixed cells in agarose were washed with distilled water three times and stained in 0.5% uranyl acetate overnight before dehydration in ascending grades of ethanol (30%-100%). Samples were transitioned from ethanol to infiltration with propylene oxide and embedded in Embed-812 hard resin (Electron Microscopy Sciences, Hatfield, PA). Blocks were sectioned at 50–60 ηm and stained with uranyl acetate and Reynolds’ lead citrate. Images were collected using a Hitachi H-7000 transmission electron microscope.

One fascicle was harvested from the tail of a 7-month-old Sprague-Dawley rat and cut into segments approximately 10 mm long. According to a protocol specifically developed for tracking collagen fibrils in tendon using serial block-face scanning electron microscopy (SBF-SEM)18 (link), the segments were fixed with glutaraldehyde, stained with osmium tetroxide, tannic acid, and uranyl acetate, dehydrated, and embedded in EMbed 812 Hard resin (Electron Microscopy Sciences). The resin block was positioned with the fascicle axis oriented vertically and then faced to expose the tissue cross-section. At a single location, 88 serial cross-sectional images (2000 × 2000 pixels, 5 nm/pixel) were taken every 100 nm along the fascicle length with a Merlin VP Compact scanning electron microscope (Zeiss) fitted with a 3View2XP in situ ultramicrotome (Gatan). The raw images were processed using Fiji27 (link) and the fibrils were tracked with a custom Matlab algorithm (see Supplementary Methods).