Lab-Tek Chamber Slide for EM Prep: New Protocols for in situ Ultrastructural Study of Monolayer Cultures Missy Hazen and Gang Ning Electron Microscopy Facility in the Huck Institutes of the Life Sciences, The Pennsylvania State University, University Park, PA 16802 Embedding monolayer cell cultures in resin for TEM usually requires scraping cells from the culture substrate, a procedure which disturbs the monolayer and often results in mechanical trauma to the cells in addition to the loss of cellular orientation and information on cellular interaction. In an alternative method monolayers are directly processed and embedded on cover slips or Petri dishes, however, one frequently encounters difficulties separating the embedded cells from the substrate. We have recently developed a new protocol in which Lab-Tek chamber slides were used to embed monolayers in epoxy and LR White resin. The current protocol is simpler and less time- consuming than conventional methods and existing protocol using Lab-Tek Chamber and the embedded monolayer can be easily separated from support substrate simply by peeling off the slide from the chambers. Furthermore, we extended using Lab-Tek chamber slides to SEM and obtained results comparable, if not superior, to cover slips and Petri dishes. Cells cultured in Lab-Tek plastic Permanox TM slides of 2-, 4-, and 8-chamber (PGC Scientifics, Cat. No. 81-6669-18, 81-6669-21, or 81-6669-24; also available via Electron Microscopy Sciences) were used. Protocol for epoxy resin embedding is as follows: 1) Rinse the fixed cells in 0.1 M cacodylate, pH7.4 and post-fix cells with 1% OsO 4 for 30 min; 2) Rinse 3x in buffer, 2x in H 2 O, en bloc stain in 2% aqueous UA for 30 min in dark; 3) 2x in H 2 O and dehydrate in ethanol (70%, 90%, and two times 100%) followed by two changes in 100% acetonitrile, 5 min each; 4) Infiltrate cells with 1:1, acetonitrile:Eponate12/Aradite resin (Ted Pella; The formula of the epoxy resin is 76g Eponate 12, 18g Araldite 502, 39g DDSA, 61g NMA, and 2-3.5% DMP-30) for 1 hr followed by 2x in 100% resin of same formulation, 1 hr each; 5) Add fresh resin up to a little more than half of the well and cure overnight in a 70°C oven; 6) Peel off the slide from chambers (Fig. 1c) and remove the plastic sides. The resin blocks are ready for sectioning. The following protocol was used to embed cells in LR White : Do Steps 1-4 on ice. 1) Quench the fixed cells with 0.15% glycine in 0.1 M phosphate buffer, pH7.4 for 5 min, then rinse 3x with the same buffer; 2) Dehydrate monolayer with 50%, 70%, 90%, and 100% ethanol, 5 min each; 3) 1:1 mixture of 100% ethanol: 100% LR White for 40 min followed by two changes of 100% LR White, 40 min each; 4) Fill up 2/3 of the wells with 100% LR White and put the lid on the chambers; 5) Polymerize at 0-4°C with UV light for 3 days or at 64°C for 24 hrs. SEM Protocol : 1) Rinse, post-fix and dehydrate the cells with attached chambers. Prior to drying, remove chambers and cut the slide to fit critical point dryer baskets; 2) CPD; 3) Mount and Sputter coat. The morphology of the cells was in good quality for analysis. The extracellular matrix shown in Fig. b was well penetrated by resin and there were no difficulties sectioning the block. Although the majority of the sections in our study were made in a horizontal/en face direction (Fig. a, b, and d), we noticed that the vertical/cross sections showed intact plasma membrane along basal side of the cells, demonstrating that separation of the chambers from slide did not cause any tearing of the cells (Fig. c). In LR White embedded samples, the resin was completely polymerized and easy to section. Microsc Microanal 14(Suppl 2), 2008 Copyright 2008 Microscopy Society of America 706 DOI: 10.1017/S1431927608082937