IEM Protocol: liver and spleen from arsenic treated mice.
1. Fix cells in 4% paraformaldehyde (PF)/ 1%GTA in PBS for 2 hours, RT. Western blot test shows IEM label still good with 1% GTA this added. Samples were fixed overnight. 3-22-05 samples were way too big. @7mmx2mmx4mm. I cut each down to 1mmx1mmx2mm.
2. Quench free aldehydes with 50mM glycine in 0.1M NaCac buffer pH 7.2
3. Rinse in 0.1M NaCac buffer pH 7.2 for 15 min.
4. Dehydration/Infiltration/Polymerization: (Newman pp. 55-65; pp. 87-99).
Dehydrate: 50% ETOH - 5 min and 70%ETOH - 2 changes, 5 min each
5. LR White: 85%ETOH - 2:1 4 changes 30 min each – can leave overnight to get better infiltration of liver tissue (overnight with 3-22-05 prep)
6. LR White - 3 changes 30-60 min each, on a gentle Rotamix.
7. Overnight in fridge (@4ºC), on a gentle Rotamix. Or go right to step 8.
8. LR white - 1 change 1 hour at RT
9. Place tissue in gelatin capsules, add fresh LR White; seal capsules; allow to settle for 1 hour.
10. Polymerize at 50ºC for 24 hours.
NOTE: if you need to centrifuge material and you have a small sample size, place cells in trimmed beam capsule, seal and centrifuge inside a 12-15ml centrifuge tube with a cotton bottom pad, using a clinical centrifuge. Then carefully seal beam capsule inside two 000 gelatin bottoms, which have been filled with LR White. Make sure you seal well with LR White. Use regular mold holders to hold capsules. OR if you need to centrifuge material and you have a large sample size, leave cells in original 00-gelatin capsule (i.e. complete step 8). Then place 00 capsule inside a BEEM capsule that has been cut to accommodate 00 gelatin capsule (i.e. top half is removed); centrifuge inside a 12-15ml centrifuge tube with a cotton bottom pad, using a clinical centrifuge, until sample is spun down.
a. Use glass vials to mix.
b. mix solns. at RT
c. keep out of direct light
d. Use a Rotamix, as solution is not very miscible & needs to be GENTLY stirred.
e. Keep Oxygen out of LRWhite &avoid excess air bubbles in resin. The dehydration and embedding are fairly straightforward.
f. Test one block for sectioning quality. Polymerize another hour, if necessary.
g. LR White blocks should be stored at -20ºC to preserve antigenicity indefinitely. Remove the gelatin and expose block to air in order to stop polymerization. (Newmann, p.104.)
h. FOR LIGHT STAIN, to see 5nm GOLD PARTICLES, use 2%UA-aq for 10 min
i. FOR STAIN, to see 12nm GOLD PARTICLES, use 2%UA-aq for 10 min and then 10 min with Reymold’s LC.
References:
Griffiths, G., Fine Structure Immunocytochemistry, Springer-Verlag, 1993
Guthrie, C. and G.R. Fink, "Guide to Yeast Genetics and Molecular Biology, V194 in Methods in Enzymology, Academic press, 1991, Chapters 41&42.
Hyatt, A.D. "Immunogold Labeling Techniques", Chapter 4 in Electron Microscopy in Biology, Harris, J.R (ed.), IRL Press, 1991
Newman, G.R. and H.A. Hobot., Resin Microscopy and On-Section Immunocytochemistry, Springer-Verlag, 1993