Immuno-EM Protocol for Ultrastructural Localization

Immuno‐EM was performed as described previously (Storrie et al. 1998 (link)). Day 7 PF were plated onto T‐25 cell flasks, washed with 4°C PBS, and scraped from the bottom of the flask. Cells were pelleted by centrifuging at 2500 g for 10 min at 4°C, resuspended in 4% gluteraldehyde, and incubated for 1 h. Cells were then pelleted by centrifugation at 2500 g for 10 min at 4°C, rinsed with PBS, resuspended in 1% OsO₄, incubated for 1 h, and rinsed with ddH₂O. Cells were then serially dehydrated by incubating for 10 min in 70% ethanol, for 10 min in 80 ethanol, for 10 min in 90% ethanol and finally for 10 min in 100% ethanol two times. Cells were then incubated with propylene oxide for 5 min three times. Cells were then embedded with 2:1 propylene oxide:epoxy resin, 1:1 propylene oxide:epoxy resin, 1:2 propylene oxide:epoxy resin then pure epoxy resin for 30 min. A vacuum was pulled for 10 min at room temperature then the resin was cured at 60°C overnight. Samples were sectioned with microtome and imaged with an electron microscope. Immunolabeling was performed using anti‐MMP (1:100) and gold‐conjugated anti‐rabbit secondary antibodies (1:100). After immunolabeling, sections were positively stained and embedded with 2% methyl cellulose containing 0.3% uranyl acetate air‐dried, and viewed using an FEI Tecnai F20 200 keV microscope (Hillsboro, OR).