Immuno electron microscopy （pre-embedding）method
１ Cutting out of tissue
２ The tissue is cut in detail. ５ｍｍＸ５ｍｍＸ２ｍｍ
３ 2.5%glutaraldehyde fixation (2 hours or more in the temperature of 4℃
are fixed. )
４ It washes it with the phosphate buffer that adds the saccharose.
①Phosphate buffer that added 10% saccharose（４℃、４hores～overnight）
②Phosphate buffer that added 20% saccharose（４℃、４hores～overnight）
③Phosphate buffer that added 30% saccharose（４℃、４hores～overnight）
５ The tissue is buried under the OCT compound, and it freezes with the acetone
dry ice.
６ The frozen section of 6 microns is stuck on making silan coated glass.
７ The cut is dried. （Room temperature at ３０ minutes）
８ It washes it with cooled PBS.
９ It washes it with 0.1% semicarbazide hydrochloric acid solution. (It
washes it once.)
１０ The aldehyde reaction is obstructed by using 0.1% semicarbazid hydrochloric
acid solution. (Room temperature for 1 hour)
１１ It washes it with the phosphate buffer solution.
１２ It washes it by distilled water.
１３ It washes it with 200 time Immunosaver dilution solution.
１４ It puts on the constant temperature machine of soak 70℃ and the antigen
retieval 16 hour processing is done to 200 time Immunosaver dilution solution.
１５ It washes it with the phosphate buffer solution.
１６ Immuno staining(LSAB）
１７ It washes it with cooled PBS.
１８ It post-fixes by 1% osmium acid. ４℃ ２hours
１９ It washes it with the phosphate buffer solution.
２０ Dehydration
２１ It embeded it in epoxy resin.
２２ Ultra thin section Electron microscope observation Electron micrograph