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TEM PREP Tissue Culture Cells (Mast cells)

for Ruth Craig

Reference:
Craig, R. etal (1998) Journal of the American Society of Hematology Vol 92 #9 p.3226

Spin cells/pellet 7 minutes at 500-1000g, maximum, for each step.

(600RPM on our clinical centrifuge for 5 minutes)

MAKE SURE CELLS ARE IN POLYPROPYLENE TUBES, NOT POLYSTYRENE

1. Pre-rinse cells two times in 0.1 M phosphate buffer, with 4% Sucrose, pH 7.4. Fix in 2% GTA/1% PF in 0.1 M phosphate buffer pH 7.4, starting at RT; leave for 15 minutes and then replace with fresh fixative, chilled to 4 C. Fix at 4 C for one hour.

2. Wash in 0.1 M phosphate buffer pH 7.4; 3X 15 mins each.

3. Postfix in 1% OsO4 in 0.1 M phosphate buffer pH 7.4 for 1hr. at RT.

4. Rinse in 0.1 M phosphate buffer pH 7.4, 2X 5 minutes each.

5. Rinse in dH2O.

6. Dehydrate in Ethanol (made from 100% ETOH) 30%, 50%, 70%, 95% - 10 mins each

7. 100% ETOH 3X, 10 mins each.

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

9. Pellet is immersed in LX112:PO - 1.5:1 {10.2 ml:6.8 ml}. Cap vials for 2 hrs. Remove caps and place in vacuum dessicator overnight.

10. Transfer pellet to BEEM capsules and fill with fresh LX112. Spin down 30 minutes at 1,000 rpm to get cells to bottom of capsule. Place in vacuum dessicator overnight.

11. Polymerize at 60 oC for 48 hrs.

Remove blocks from heat to cool.

12. Section: 70-90nm; stain: UA MeOH for 20'; LC for 5' .

Last Updated: 10/2/08