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TEM PREP Growth plate vasculated interface of 6 week old mice

For Roy Fava; VA Hospital (Medicine DHMC)

Hunter, W.L. and Arsenault, A.L. 1990 "Vascular Invasion of Epiphyseal growth plate: Growing Metphyseal Capillaries", Anat. Rec 227:223-231

1. Excise 1mm3 tissue. In the future, I told Dr. Fava he could cut out a larger piece, fix for 15 minutes and then cut done after tissue "toughened" in fixative. Fix in 4% GTA 0.1 M Pipes buffer pH 7.4, at RT; leave for 15 minutes and then replace with fresh fixative. Fix 24hours.

NOTE: 8/98 prep. - Pipes buffer does not preserve plasma membrane as well as I would like, although inner ultrastructure is wonderful; so: all future preparations are use 3%GTA/2%PF in 0.1M NaCac pH 7.4 (like I used for mouse embryos), for better membrane preservation.

9/9/98: 3%GTA/2%PF in 0.1M NaCac pH 7.4 fix for 24 hrs.

2. Tissue was demineralized in 4% EDTA in Tris buffer(300mOsm, pH 7.3) at 4oC for 4 days with twice changes of solution daily.

3. Rinse three times in 0.1M NaCac buffer pH 7.4 .

4. Postfix in 2% OsO4 in 0.1M NaCac buffer pH 7.4 for 2hr. at RT.

5. Rinse in dH2O.

6. Dehydrate in Ethanol(made from 100% ETOH) 30%, 50%, 70%, - 10 mins each

(leave overnight in 70% ETOH)

7. 100% ETOH 4X, 10 mins each.

8. Two 10 min. changed in Propylene oxide(PO).

9. Tissue is immersed in LX112:PO - 1.5:1 {10.2 ml:6.8 ml}. Cap vials for 6-7 hrs. Remove caps and place in vacuum desiccator overnight.

10. Transfer Tissue to Flat embed mold and fill with fresh LX112. Mount with razor cut side facing the block end. This should be growth plate end. Place in vacuum desiccator 24 hours.

11. Polymerize at 60 oC for 48 hrs.

Remove blocks from heat to cool.

12. Section: 100-120nm - use glass knives because of presence of calcified tissue; stain: UA MeOH for 20'; LC for 5' .

Last Updated: 10/2/08