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

Start-End/Date	Solution/ notes	Duration/Temperature
	Wash in 0.15 M Cac	30 min RT
	2% OsO4 in Cacodylate (0.15M) Note: Prepare TCH	90 min RT
	2,5% Ferrocyanide in Cacodylate (0.15M)	90 min RT
	2% OsO4 in Cacodylate (0.15M)	45 min RT
	Wash in 0.15 M Cac	30 min RT
	1X Rinse in Water	30 min RT
	Saturated Thiocarbohydrazide (RT)	60 min, RT
	2X Rinse	30 min
	2% OsO4 aqueous solution Note: Prepare UA	90 min, RT
	2X Rinse	30 min
	2% uranyl acetate aqueous solution	Overnight, 4°C
	Next day warmed up in oven (in uranyl sol)	to 50 °C for 120 min
	2X Rinse	30 min
	Lead Aspartate solution (0.066 g Lead nitrate in 10 ml 0.03M Aspartic acid, pH adjusted to 5.0 with KOH)	50°C for 120 min
	2X Rinse	30 min
	50% ethanol	30 min 4°C
	75% ethanol	45 min 4°C
	100% ethanol	45 min RT
	3X Pure acetone	45 min each RT
	1:1 mixture of acetone: complete spurs resin (all 4 components) CLOSED caps	3 hr at RT (no rotation)
	Acetone evaporation: Open the caps and put on the rotator	90 min rotation + ON
	Replace old resin for fresh CLOSED tubes	6hr at RT
	Transfer to mold and move to oven	At least 48 hr at 70 °C

Solutions:

2% OsO₄in Cac: Mix 4% OsO₄Solution 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!