Processing Platelets for Standard TEM

Protocol for EM of Platelets

SAMPLE COLLECTION:
1. Anesthesia of mice w/ (Ketamine 4uL, Anased 1uL) 2-2.5 uL/g (body weight)
2. Collect blood by closed-chest cardiac puncture -> 1cc syringe w/ 0.15 mL ACD 23-25-gauge needle
3. Rinse 15 mL Falcon tube with ACD and put collected blood in tube
4. Add 2X vol. Tyrode solution
5. Transfer into 2.0 mL tubes, centrifuge @ 300g at RT for 5 min
6. Put supernatant into 2.0 mL Eppendorf tube

FIXATION:
1. Add an amount equal in volume to the platelet suspension of 0.2% Gluteraldehyde in White’s Saline, incubate 20min @ 37 oC
2. Pellet platelets @ 15,000g for 5 min. @25 oC in a benchtop microcentrifuge
3. Fixe the pellet with 3% Gluteraldehyde in White’s Saline @4 oC for 2hr or overnight

WASH:
3x15 min. w/ White’s Saline @4 oC

OSMIFICATION:
1% OsO4 in WBS 1.5 hrs. @4 oC

WASH:
2x10 min. w/ ddH2O @4 oC

URANYL ACETATE:
3% UO2 in ddH2O 1 hr. @4 oC

WASH:
2x10 min. w/ ddH2O @4 oC

DEHYDRATION:
• @4 oC, 30%, 50%, 70% ETOH, carefully get the pellet off the wall using angled wood sticks (freshly cut with razor blade)
• Change to room temperature from 85%, 95%, 99.5% 1x15 min.
• 100 (200 Proof) % 2x15 min
• Propylene Oxide 2x15 min RT

INFILTRATION:
• PO : Araldite/Epon = 1:1 for 1 hr. RT
• PO : Araldite/Epon = 1:2 overnight RT

EMBEDMENT:
• (Accelerator added to Resin) Pure Resin 1 hr. RT
• Embed into blocks and cure overnight @45 oC and 48hrs. @60 oC

Platelet Resin

White's Buffer Solution

White’s Buffer Solution

White’s Solution A
Dissolve 14g NaCl (2.4M), 0.75g KCl (0.1M), 0.55g MgSO4 (46mM), 1.5g Ca(NO3)2.4H2O (64mM) in 100 mL ddH2O, and correct pH to 7.4. Store @ 4 oC

White’s Solution B
Dissolve 1.1g NaHCO3 (0.13M), 0.22 g Na2HPO4.7H2O (8.4mM), 0.05g anhydrous KH2PO4 (3.8mM), 0.01g phenol red in 100mL ddH2O, and correct pH to 7.4. Store @ 4 oC.

Glutaraldehyde in White's Saline