2% GA in 0.05M PB, 0.1M Sucrose

2.5% GA, 4% PFA in 0.1M CB, 0.1M Sucrose

5% Gluteraldehyde in 0.1M Cacodylate Buffer

1% OsO4 in buffer

2% OsO4 in buffer

2% GA, 2% PFA in 0.1M Cacodylate Buffer

2% GA, 2% PFA in 0.1M Phosphate Buffer

Perfustion Fixative II

Making 200 mL of fixative for perfusion (2.5% GA, 4% PFA in 0.1M Phosphate Buffer)

• Heat 120 mL ddH2O to >60C<85C (do not boil)
• Add 8 g powdered Paraformaldehyde and immediately cover flask with foil to avoid toxic vapors (prepare in fume hood if possible)
• Clear with NaOH (one drop at a time, perhaps only one is necessary)
• Cool to RT with an ice bath
• Add 20mL of 25% Gluteraldehyde
• Add 50 mL 0.4M Phosphate Buffer
• QS to 200 mL with ddH2O
• Filter through a hydrophilic filter and aliquot into 50 mL tubes.
• Check pH and adjust to 7.2-7.4 with 1M HCl
• Store at -20C

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.

3% Paraformaldehyde for IEM

0.1M Cacodylate Buffer containing 4% Sucrose and 0.1M Glycine IEM

0.1M Cacodylate Buffer containing 4% Sucrose IEM

Making Urynal Acetate and Lead Citrate

Reynolds lead citrate

Place the following components into a volumetric flask:

1.33 g Lead nitrate
1.76 g sodium citrate
30 ml ddH2O

• Shake vigorously for 1 min and then intermittently for the next 30 minutes. Add 8 ml of 1M NaOH, at which time the solution becomes clear. Use only freshly made carbonate-free NaOH solution. Add ddH2O to the 50 ml mark and mix by inversion until the precipitate is dissolved.
• The pH should be about 12. It will keep for several months if kept stoppered.
• Aliquot into 1.5 mL eppendorf tube through a hydrophobic filter.
• Centrifuge @12k RPM for 3.5 min. before staining.

Uranyl Acetate (3% in 50% Methanol)

Can be made in a 15 mL tube.

0.42 g UA
14 mL 50% Methanol

• Mix and dissolve on a rotator 30 min.
• Store for 2 days @4C before use
• Aliquot into 1.5 mL eppendorf tube through a hydrophilic filter.
• Centrifuge @12k RPM for 3.5 min. before staining.

Postembedding Immunolabeling

Postembedding Immunolabeling

1. Cut sections (>100 nm thick) on formvar coated Ni grids

2. Labeling on drops
a. 1x5 min. with 0.1MPBS (pH7.4) containing
i. 1%BSA
ii. 0.05% Triton x-100
iii. 0.05% Tween 20

b. 2 hr. @ RT and @4C overnight with Antibody in 0.01M PBS (pH7.4) containing
i. 1%BSA
ii. 0.05% Tween 20

c. Wash 3x1 min. on drops of 0.01M PBS

d. 1 Hr. @RT Incubation with secondary antibody conjugated with Au labels in 0.1M PBS containing
i. 1% BSA
ii. 0.05% Tween 20

e. Wash 3x1 min. on drops of 0.01M PBS

f. Allow grids to dry

3. Counter stain
a. 3% UA 4 min.
b. Lead citrate <1min.

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

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

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.

0.18M Phosphate Buffer (muscle)

Isoosmotic Phosphate Buffer (Muscle)

0.1M Tris Wash (Muscle)

0.4M Phosphate buffer Stock (Muscle)

Mouse Glutaraldehyde Fixative (muscle)

1% OsO4 in 0.1M PB for Muscle

Toluidine Blue Stain

Materials:
Sodium Borate Tetra (EMS, CAT #21130)
Toludine Blue (EMS, Cat # 22050)
ddH20
150 mL beaker
Magnetic stir plate and bar
Hydrophylic filter

1. Wrap beaker with metal foil and combine the follow ingredients in order in a 150 mL beaker:
a) 1 g Sodium Borate Tetra
b) 100mL ddH2O
c) Stir until dissolved
d) 0.5-0.7 g Toludine Blue
e) Stir until dissolved (can be allowed to mix o/n), cover top so as to not let any light in

2. Filter prior to use
3. Store @RT

Sodium Phosphate Buffer from Monobasic and Dibasic Stock

Here is a quick table for mixing up 0.1M Sodium Phosphate buffer from Monobasic and Dibasic stock solutions. To make it 0.2M, don't add water. Enjoy!

Processing tissue for Immuno Electron Microscopy

Again buffers and fixative percentages can vary from sample to sample but here's my protocol:

01) Fixation 2hrs. @RT and o/n @4C

02) Wash with buffer containing 4% sucrose 4x15 min. @4C

03) Wash with buffer containing 4% sucrose and 0.1M Glycine 1x30 min. @4C

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

05) Infiltration with Lowicryl LK4M
Methanol:LK4M = 1:1 1 hr. @-30C
Methanol:LK4M = 1:2 1 hr. @-30C
Methanol:LK4M = 1:3 1 hr. @-30C
Pure LK4M 1 hour @-30C
Pure LK4M o/n @-20C

06) Embed in Beem capsules @-35C with dry nitrogen bubbling in Lowicryl

07) Cure under UV light in a dry ice environment @-35C 36hrs. and @RT for 12 hrs

Heavy Metal Staining for TEM Thin Sections

Here is a protocol for staining thin sections for transmission electron microscopy:

Staining

Set up a moisture chamber:
• In an inverted 60 mm petridish place a 100 mm disk of filter paper and wet with ddH2O
• Cut a piece of parafilm to fit and place it on the wet filter paper in the dish.

Set of ddH2O wash:
• Cut a piece of parafilm
• Wet surface of bench with water
• Place film on water to stick to bench
• Place 100 uL drops of ddH2O on the film in an array of 3 x n where n = the number of grids to be stained

Staining:

UA:
• Place grid on a 5 uL drop of UA in a moisture chamber
• Cover with lid and place chamber in the dark or cover with metal foil to avoid light exposure to the uranium (light can cause U precipitate on section)
• Leave for 20 min.
• Pick up grid with tweezers and tap off remaining UA
• Immediately place onto the first 100 uL drop of ddH2O
• After 2 sec. move grid to second 100 uL drop and let sit for about 1 min.
• Transfer to the third drop for another minute
• Pick up grid with tweezers and tap off remaining water
• Transfer to grid box and allow grid to dry for several minutes

Pb:
• Cut a new piece of parafilm for the moisture chamber and place 4 or 5 pieces of Sodium Hydroxide around the edges and cover
• Place grid on 5 uL drop of lead citrate while avoiding any exhalation of breath(CO2 can cause Pb precipitate on section)
• Leave for no more than 5 min.
• Pick up grid with tweezers and tap off remaining Pb
• Immediately place onto the first 100 uL drop of ddH2O (again, be careful not to breath on the grid)
• After 2 sec. move grid to second 100 uL drop and let sit for about 1 min.
• Transfer to the third drop for another minute
• Pick up grid with tweezers and tap off remaining water
• Transfer to grid box and allow grid to dry for several minutes

Making a loop for grid transfer

Here is an instructional video for making a loop to transfer grids from one drop to another. In the video, they drill a hole in the applicator stick to put the loop into, but it is easier to just whittle the stick to a point and use a 20 ul pipette tip to hold it in place.

Making Resins

Here are some handy tables for mixing up various resins. Most people measure them out in weight, but we've found it easier to measure by volume. Lowicryl must be mixed with dry nitrogen bubbles and stored at -20C. Click to enlarge.

Sodium Cacodylate Buffer

0.4M Sodium Cacodylate Buffer (250 mL)

0.4M Sodium Cacodylate = 21.4 g / 250 mL ddH2O

Weigh out 21.4 g Sodium Cacodylate and add to 200 mL H2O. Mix with a magnetic stirring rod. Adding about 8 mL of 0.2M HCl should adjust the pH to 7.2-7.4, however adding stronger HCl drop-by-drop and checking pH will also work. A target pH of 7.4 is better since the pH can lower slightly throughout processing. QS to 250 mL with ddH2O.

Perfusion Fixative I

Making 200 mL of fixative for perfusion (1% GA, 4% PFA in 0.1M Phosphate Buffer)

• Heat 120 mL ddH2O to >60C<85C (do not boil)
• Add 8 g powdered Paraformaldehyde and immediately cover flask with foil to avoid toxic vapors (prepare in fume hood if possible)
• Clear with NaOH (one drop at a time, perhaps only one is necessary)
• Cool to RT with an ice bath
• Add 8mL of 25% Gluteraldehyde
• Add 50 mL 0.4M Phosphate Buffer
• QS to 200 mL with ddH2O
• Filter through a hydrophilic filter and aliquot into 50 mL tubes.
• Check pH and adjust to 7.2-7.4 with 1M HCl
• Store at -20C

Note: This fixative was designed to preserve muscle tissue. This protocol will also work with other buffers (PBS or Cacodylate) and with different percentages of fixative. Many times 2.5% GA is more appropriate.