Processing tissue for Immuno Electron Microscopy

Again buffers and fixative percentages can vary from sample to sample but here's my protocol:

01) Fixation 2hrs. @RT and o/n @4C

02) Wash with buffer containing 4% sucrose 4x15 min. @4C

03) Wash with buffer containing 4% sucrose and 0.1M Glycine 1x30 min. @4C

04) Dehydration with Methanol:
30% 1x15 min. @4C
50% 1x15 min. @4C
70% 1x15 min. @-10C
85% 1x15 min. @-20C
95% 1x15 min. @-20C
100% 3x15 min. @-30C

05) Infiltration with Lowicryl LK4M
Methanol:LK4M = 1:1 1 hr. @-30C
Methanol:LK4M = 1:2 1 hr. @-30C
Methanol:LK4M = 1:3 1 hr. @-30C
Pure LK4M 1 hour @-30C
Pure LK4M o/n @-20C

06) Embed in Beem capsules @-35C with dry nitrogen bubbling in Lowicryl

07) Cure under UV light in a dry ice environment @-35C 36hrs. and @RT for 12 hrs