Hi David,

if you do not have the possibility to proceed your paraffin embedding in an embedding automate, then you can do it completely by hand. You need:

embedding cassettes, wide neck containers which you can close , forceps to transfer the cassettes form one container into the other, alocohol of several delutions, Isopropanol and Xylene and a 60°C oven where you melt your paraffin and do the final praffin steps. That's all ! Instead of succrose kryoprotections you can leave the tissue blocks in PBS until you start your embedding process.

Ute Neubacher Thank you for your answer. In particular, I was looking for actual links to products, like what do people put the cassettes in when transferring through the solutions during the tissue processing phase? What containers do you do the dehydration in? What oven do you recommend?

I agree with Ute's answer. Keep it simple. I use beakers as my wide neck containers. When there are volatile liquids in them (ethanol, xylene, etc.), I cover them with a glass Petri dish top. Forceps to transfer cassettes are a must. We have a vacuum oven for the paraffin steps so that we don't need to worry about xylene fumes. If you are concerned about handling xylene then you can switch to a safer clearing agent (I like Clear-Rite 3); it takes longer to process (~1 hr per step) but is easier to handle.

The only special equipment we use is for the staining. You also don't need an automated machine for this. You can get a slide staining rack or some Coplin jars.

I hope that helps. Good luck!

- Melissa

Melissa Chernick Thank you for the answer. Now I understand you move the cassettes one at a time with forceps. I was thinking of using a strainer and moving the strainer with multiple cassettes from beaker to beaker. It would probably be messy in a strainer. Thanks for the tip on the clearing agent. I do think I'll go with an alternative to xylene.

New Question, do use agitation while the cassettes are in each solution, like a rocker?

I think that a strainer would be very messy. It does not take long at all to move cassettes with forceps. Also, make sure that you write on the cassettes using a pencil or ethanol resistant lab marker or else all of your labels will disappear.

I do not agitate the cassettes. Just make sure the cassettes are fully submerged in the container. To that point, when moving cassettes to the next container, make sure to let the liquid drain out of the cassette before moving it over or you will change the concentration of your next step. It doesn't/shouldn't be completely dry, just make sure most of the liquid is out.

Hi DAvid,

I have add 2 images of wide neck bottles and glas boxes which are suitable for embedding processes. To garantee a slidely shaking of the solutions you can put the bottle or the box on a rocking table as you can see it in the imgages. With the forceps you can tranasfer the cassettes easily from one both to the next one.

If you like tu use xylene as an intermedium, do it under a fume hood. The paraffin step you can do in a oven without vacuum facilities. In that case you need to stay longer in the paraffin bath e.g first paraffin over night second paraffin 3 -4 hours or longer. This depends of the size of your tissue blocks .

If you want to process the sample manually, what you need are glass vials and thermostatically controlled embedding oven. The dehydration and clearing will be done in glass vials. The infiltration will also be carried out in glass vials placed in the oven. These two instruments/ equipment can easily be procured.

Melissa Chernick Thanks again, I finally got my head around how you are doing this with beakers and forceps. Ute Neubacher thanks for the photos! I have used those dishes for staining slides and they will be perfect for dehydration and presumably they are oven safe. I have heard slight rocking can be good but also heard brain is particularly fragile during processing. I will probably either try without or with minimal rocking.

Akinwumi Johnson Akinloye Thank you.

The oven stage is is probably the most novel step for me.

I have this protocol to use as a starting point from ICH World.

The mouse brain samples will start off having been fully fixed in 4% Formalin and trimmed to (unsure) 5mm

a. 70% Ethanol, two changes, 1 hour each

b. 80% Ethanol, one change, 1 hour

c. 95% Ethanol, one change, 1 hour

d. 100% Ethanol, three changes, 1.5 hour each

e. Xylene or xylene substitute (i.e. Clear Rite 3), three changes, 1.5 hour each

f. Paraffin wax (58‐60 ºC), two changes, 2 hours each

g. Embedding tissues into paraffin blocks, I actually have an embedding station for this so.. excited to give it a try :)

I need to do a bit more research to find the proper length of each step. Do any of you work with brain tissue?

Steps a-e at room temp. For the wax steps.. I am picturing melting the wax in the oven then simply dropping the samples in.... is it that simple?

I have a budget for setting up this lab and can probably get a vacuum oven if that has significant advantages. Any oven recommendations?

Hi David, your embedding protocol is fine. For the paraffin step: use 2 containers with melted praffin, because I would recommand to change the paraffin onece a time. You can stay in the first paraffin overnight and 2-3 hours in the second paraffin and after that you should prepare the blocks. Ich you have a vacuum oven the duration in paraffin could be decreased. In that case you have to found out experimentally what is the the optimal duration time for. you.

Ute Neubacher Thank you again. It looks like it will be important to know which steps can last over night :) does that mean the tissue is in the oven overnight?

If you decide to use xylene then your timing can be 20-30min per step. For Clear Rite 3, you will want 60-90 min per step. If you have thick/large tissue, then you may consider extending the time for Clear Rite 3 step 1 up to 3-5 hours. And yes, using these chemicals in a chemical fume hood is best, and make sure you wear appropriate PPE.

For the wax steps, you can keep the tissue in the cassette and move it over into the melted wax with forceps, as you do in other steps. You need to keep the wax at a steady temperature so that it stays molten. We keep ours at 58C. Embedding stations are nice so glad to hear you have one of those.

Our vacuum oven is made by a company called Lindberg/Blue and is sold through companies like ThermoFisher Scientific. It has worked very well for many years.

I use 2 overnight steps. The first is final steps in the ethanol dehydration --> abs EtOH for 1) 1 hour, 2) overnight, 3) 1 hour (each of these steps is a new beaker of EtOH. It may be a bit of overkill but I really like to make sure that all of the water is out of the tissues. The next is the paraffin steps. I agree with Ute's answer on this. I use 3 beakers with melted paraffin (2 is fine though) and do the overnight step as either the first or second step. But as Ute said, you can decrease that time dramatically if you have a vacuum oven, as little as 1 hour per step depending on size of tissue. You just want to make sure you have complete infiltration of the paraffin into the tissues or they will not section properly.

Melissa Chernick Thank you for that informative reply.

You can stay in the paraffin overnight. That is not a problem. To get a more or less complete dehydration I recommand to stay in Isopropnol before you go into xylene or other intermedia. I f you do not have any isorpropanol , fesh 100% ETOH is fine.

Ute Neubacher Thank you for your input. Are you saying you have had better results using Isopropanol than Ethanol? I don't have either so will be ordering what's needed. I can probably get both. In the past I have used Ethanol.

Matthew Ibbs I really appreciate your comments and advice. I think a tissue processor is not out of the question in the future. For now, my goal is to keep the budget low and demonstrate that we can get good/useful images. When this is demonstrated, not only will I know more about the process, but I will be able to make a better case for more spending. I appreciate the 3mm guidance on thickness because I have been debating how thick to make the blocks. I also appreciate the information on trouble shooting. When something goes wrong, its good to have some idea as to why!