En-bloc sample preparation for 3D electron microscopy

We use the same fixative solution for perfusion and overnight fixation.

I have attached two documents for the protocols used in that publication. One is a full description and the other a shorthand checklist. They follow the description in the methods.

I would also recommend looking at the two publications that describe the staining methods developed in our lab in more detail:

Hua et al 2015 (https://www.nature.com/articles/ncomms8923)

Song et al 2023 (https://www.nature.com/articles/s41592-023-01866-3)

Check List EM Staining

Solutions:

• 2% OsO₄ in Cac: Mix 4% OsO₄ Solution with 0.3M Solution of Cac in a 1:1 proportion. e.g. 2 ml OsO₄ with 2 ml of 0.3M Cac
• Saturated TCH: Make a 1% mixture (e.g. 0.1g of TCH in 10 ml of nanopure water). It takes a long time to get dissolved so prepare it right after you have prepared the first solution and put it on rotater at a low speed (10-13 rpm).
  ***TCH is light sensitive and is oxidized when in contact with air. Therefore, cover it with Alu foil and do not prepare it in a large container with a lot of air space
  *** light sensitive
• ferrocynide: 2.5% solution in 0.15 M Cac buffer (e.g. 0.5g in 20 ml of 0.15 M Cac) ***Light sensitive
• 2% OsO₄: 1:1 mixture with nanopure water
• Uranyl Acetate: 2% solution in nH₂O (e.g. 0.4 g UA in 20 ml of nH₂O)***Uranyl acetate is light sensitive
• Aspartate buffer: 0.03 M in  nH₂O pH adjusted to 3.8 (e.g. 2g of aspartic acid in 500ml of nH₂O)
• Lead Aspartate: for 0.066g of lead nitrate add 10ml of Aspartate buffer then adjust the pH to 5.0 with 1N KOH *** light sensitive
• Spurr' resin: 4.1 g ERL 4221, 0.95 g DER 736, 5.9 g NSA, 0.1 g DMAE (113 ul)
   

EM staining Protocol week of 25.01.2016: 

• This is the incubation step

• This is solution making guide
  ***This is light sensitivity alert

-This is general guidance

- For all the steps, we used a timer and started simultaneously with the change of solution in the first (eppendorf) tube. This would help the consistency of timing.

-Let the tubes lie horizontally so that you would have a less pronounced gradient around sample and better staining (Do this for all the incubation steps in this protocol)

-We used freshly ordered chemicals or material from freshly opened containers for all steps. Just as a control.

• 1X rinse in 0.15 M Cac: Exchange the solution in the tube with the 0.15 Cac (no need to change the tube). 30 min at RT

• Buffered Osmium 2%: Dilute with Cac buffer (0.3 M) with 1:1 proportion (no filtering needed)

• First buffered Osmium Step: Transfer the sample to buffered Osmium 2% solution for 90 min at RT 

• Samples are sticky and sensitive (membranes are not fixed) before Osmium step. Therefore, be careful so that they would not stick to the wall of the pipette in case you are transferring them
• Aqueous 1% TCH: now is the time to also prepare TCH since it takes a long time to dissolve. (e.g. Make 0.5 g in 50 ml water). Then, Use the rotator with speed 17 rpm to help dissolve the TCH. Filter before use

***Cover the bottle with Foil since TCH is photosensitive

• You can add more TCH than the amount calculated since 1% TCH is saturated
• Don’t leave a large amount of air in the container since it would oxidize TCH
• Ferrocyanide 2.5% in Cac: make it fresh 30 min before using it (time for dissolving) using 0.15 Cac buffer (e.g. 0.5g in 20 ml 0.15M Cac buffer)
  • Use the rotator with speed 17 rpm to help dissolve the KFeCN

***Cover the bottle with Foil since ferrocyanide is photosensitive

• Ferrocynide step: Transfer the sample to KFeCN solution without washing (new tube filled with ferrocynide). Incubate for another 90 min at RT

• Second buffered Osmium Step: Transfer the sample to the Buffered 2% Osmium solution as you did for the ferrocynide step (no washing new eppi filled with Os). Incubate for 45 min at RT

• 1X rinse in 0.15 M Cac: Exchange the solution in the tube with the 0.15 Cac (no need to change the tube). 30 min at RT

• 1X rinse in Water: Transfer samples to new tubes containg water. 30 min at RT

• TCH step: Add 1 % TCH (pinkish color) to the tube after removing water. 1 hr at RT

• Washing 2X: This step is repeated several times through this protocol. First use the tube from the last step and just change the buffer to water. For the Second washing, use a new tube filled with water and transfer the sample using a pipette with a big opening (cut the thin parts). This tube will be used for your next buffer incubation step. Each wash 30 min at RT 

• Osmium 2%: mix Osmium 4% and water with a 1:1 proportion (no need to filter)

• Aqueous Osmium Step:Transfer the sample to Osmium 2% solution and incubate for 1.5hr at RT

• 2% Uranyl acetate: Make it now for the dissolving time. (e.g. 0.4 g UA in 20 ml of Water). Filter before use

***Cover the bottle with Foil since UA is photosensitive

• Washing 2X

• Uranyl acetate step: Add 2% Uranyl acetate after removing water. Leave ON at 4°C. Next morning. Transfer the samples to oven at 50°C for 2h.

• Uranyl acetate is slightly radioactive therefore use the appropriate waste container for disposal.

• Washing 2X

• Lead aspartate: Dissolve each 0.066g of lead nitrate in 10ml aspartate solution, then use KOH to set the pH between 5 and 5.1 (it is much better if it is closer to 5.00). No need to filter. This whole process would take around 30 mins

***Cover the bottle with Foil since lead aspartate is photosensitive

• Lead aspartate step: Add lead aspartate after removing water. Incubate at 50°C for 2h

• Washing 2X

-After the change during this washing step, we use the same tube during the dehydration and acetone steps. No need to change the tube.

• Dehydration 50% step: take out the entire buffer in the tube add 50% ethanol solution incubate at 4°C for 30 min. 

• Prepare the 50% mix beforehand and keep it at 4°C

• Dehydration 75% step: take out half the solution in the tube and add pure ethanol. Incubate again in the fridge at 4°C for 45 min.

• Prepare the 75% mix beforehand and keep it at 4°C

• Dehydration 100% step: Take out as much of the solution possible without DRYING OUT!! It is really important NOT to DRY OUT the sample. Then add pure ethanol and incubate at RT for 45 min

• Leave the 100% ethanol bottle outside beforehand to adjust to room temperature

• 3X acetone step:Remove the ethanol and add pure acetone (We used the acetone from Serva) (similar to last step no DRYING OUT). Incubate for 45 min at RTeach (repeated three times)

• Start preparing the resin 45 min before the end of last acetone step
• First mix the first three components. Let them mix and add the accelerator just before mixing the resin with acetone

• First infiltration step: Remove the acetone and add 2 ml per sample the 1:1 mixture of acetone and complete resin (all 4 components). for 3 hr at RT, 

• Keep the caps closed and no rotation The idea is to enhance infiltration of resin monomers with acetone around

• Second infiltration step: Open the tube caps remove half of the actone:resin mixture (leave 1ml in) and set the timer of the rotator (low speed: 6 rpm) for 90 mins and leave them overnight for acetone evaporation

• Third infiltration step: Transfer the samples to (1 ml per sample) fresh complete resin mixture (all 4 components) for 6 hrs at RT. 2 pm Friday

• Bake the samples over the weekend (72 hrs) at 70°C in the oven. Oven turned off at 4 pm Monday!