Ace200 sputter coater

Briefly, samples for SEM were fixed in 2% glutaraldehyde in cacodylate buffer, followed by rinsing in buffer and dehydration in a graded ethanol series. The samples were critical point dried in a Bal-tec CPD030 critical point dryer, mounted on aluminum stubs, gold coated in a Leica ACE200 sputter coater, and examined in an FEI XL30 SEM.

Cell-scaffold cultures were collected at various intervals and fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH7.4. For routine transmission EM samples were post-fixed in 1% osmium tetroxide, dehydrated in an ascending series of ethanol, infiltrated with propylene oxide, and embedded in Quetol-Spurr resin. Ultrathin sections were then cut with a Leica EM UC7 ultramicrotome, mounted on grids, and stained with uranyl acetate and lead citrate prior to image acquisition with a FEI Tecnai 20 TEM. For scanning EM samples were post-fixed in 1% osmium tetroxide, dehydrated in an ascending series of ethanol, critical point dried using a Bal-tec CPD030 critical point dryer, and mounted on aluminum stubs. Samples were gold-coated using a Leica ACE200 sputter coater and examined and photographed with a FEI XL30 SEM. For immunogold EM samples were processed as described previously⁷⁵ . Ultrathin sections were cut to gold thickness and placed onto 400-mesh copper grids for immunogold. Immunogold labeling was performed as previously⁷⁵ using 1:200 diluted rabbit anti-TP63 (Cell Signaling) or anti-KRT5 (Abcam) antibodies followed by a 1:300 diluted 10-nm gold-conjugated goat anti-rabbit IgG (Nanoprobes). Samples were then stained with 3% (w/v) uranyl acetate and 1% (w/v) lead citrate and examined on a Philips 430 electron microscope.