This section of pancreas was prepared by freeze substitution using a simple method.
This pancreas was fixed in buffered 4% paraformaldehyde and cryoprotected in 2.1M sucrose. Small pieces of the cryoprotected tissue were frozen by immersion in liquid nitrogen and then transferred to methanol, on dry ice, containing 1% osmium tetroxide. After 1 day, the methanol/osmium tetroxide was removed and replaced with fresh, cold methanol and left for a further day. The methanol was gradually replaced with cold Lowicryl HM23 and the block was polymerized, still on dry ice, with indirect UV light.
Thin sections were treated sequentially with antibodies to a glycoprotein (GP2) and with protein A gold. The protein A gold, visualizing the sites where antibodies are bound to the section, are seen as small round particles outside the cells on the left of the micrograph.
Antibody Labeling