Standard processing for IEM

1. Fixation:
3% PFA in 0.1M Buffer with 0.1% GA and 4% sucrose

2. Wash:
4x15 min. with correlative buffer containing 0.1M Sucrose @ 4C
1x30 min. with correlative buffer containing 0.1M Sucrose and 0.1M Glycine @4C

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

6. Infiltration with Lowicryl LK4M:
METOH:LK4M = 1:1 for 1 hr. @-30C
METOH:LK4M = 1:2 for 1 hr. @-30C
METOH:LK4M = 1:3 for 1 hr. @-30C
Pure LK4M 1 hr. @-30C
Pure LK4M overnight @-20C

7. Embedment:
Embed into Beem Capsules and cure under UV in a CO2 environment @-35C for 36 hr. and under UV in a CO2 environment for 12 hr. @ RT.

Processing hard to penetrate tissues for standard TEM

1. Fixation:
According to tissue
2. Wash:
3x15 min. with correlative buffer @ RT
3. Osmification:
1 or 2% OsO4 in correlative buffer 1-2 hours @RT
4. Wash:
3x15 min. ddH2O @RT
5. Dehydration with ETOH and Propylene Oxide @RT:
30%, 50%, 70%, 85%, 95%, 100% ETOH 1x20 min. each
200 Proof (pure) ETOH 1x20 min.
Pure PO 2x20 min.
6. Infiltration with Epon:
PO:Epon = 1:1 for 1 hr.
PO:Epon = 1:2 overnight
Pure Epon for >1hr.
7. Embedment:
Embed into blocks and cure @45C overnight and @60C 48 hr.

Standard processing for TEM

1. Fixation:
According to tissue
2. Wash:
3x15 min. with correlative buffer @ RT
3. Osmification:
1 or 2% OsO4 in correlative buffer 1-2 hours @RT
4. Wash:
3x15 min. ddH2O @RT
5. Dehydration with ETOH and Propylene Oxide @RT:
30%, 50%, 70%, 85%, 95%, 100% ETOH 1x15 min. each
200 Proof (pure) ETOH 1x15 min.
Pure PO 2x15 min.
6. Infiltration with Epon:
PO:Epon = 1:1 for 1 hr.
PO:Epon = 1:2 overnight
Pure Epon for >1hr.
7. Embedment:
Embed into blocks and cure @45C overnight and @60C 48 hr.

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

Muscle Processing for Standard TEM

Muscle protocol and flat embedment muscle protocol. Utilize a rotator or shaker if possible for all steps.

1. Fixation:
According to tissue
2. Wash:
2x15 min. 0.1M Tris buffer @RT
2x15 min. isoosmotic buffer @RT
3. Osmification:
1% OsO4 in 0.1M PB 1-2 hours @RT
4. Wash:
3x15 min. ddH2O @RT
5. Dehydration with ETOH and Propylene Oxide @RT:
30%, 50%, 70%, 85%, 95%, 100% ETOH 1x15-20 min. each
200 Proof (pure) ETOH 1x15-20 min.
Pure PO 2x15-20 min.
6. Infiltration with Epon:
PO:Epon = 1:1 for 1 hr.
PO:Epon = 1:2 overnight
Pure Epon for >1hr.
7. Embedment:
Embed into blocks and cure @45C overnight and @60C 48 hr.