Hi Yuka,

entire adult mouse brains do not need to be embedded for vibratome cutting, after formaldehyde fixation they are solid enough and you can glue them directly on the sample holder. We place a small block of 4% agarose behind the brain, just to avoid that the it is pushed back by the advancing blade.

Embryonic brains or dissected brain areas we embed in 4% low-melting agarose (Sigma A9414, in Europe we use a cheaper version, Biozym 850080), that is an agarose that gels below 35-36 °C, so you can melt it at about 65 °C, as usual, then place it in a 37 °C bath to cool down and it will remain liquid, and a this temperature (physiological temperature) you can embed the tissue in the mold and wait until it goes below 35 °C, when it gels.

We always cut in iced PBS, anyway, to have the maximal density in the tissue.

Good luck.

Hi Pietro,

I need agarose since I cut my samples into pieces already (fairly tiny).

Is there particular reason to use 4% LM agarose?

I've heard that people use 2% agar/agarose fairly often.

Kind regards,

Yuka

Well, the gelling point of standard agarose is typically above 40 °C, worse for agar, probably closer to 50 °C, which means you have to pour the material at a higher than physiological temperature on your tissue... We prefer to pour material that is around the physiological temperature of 37 °C on our brain tissue, that's why we prefer the low-melting agarose (that would be better to call low-gelling, since melting is pretty much the same...). The %age is also your choice, rigidity helps cutting the slices more uniformly and thinner; it is also easier to take off the tissue after you have cut the slice (leaving the agarose during IF staining is not recommended, it traps a lot of antibody decreasing your staining quality and giving a bright halo all around the slice during imaging), that's why we prefer 4%, but of course also 2% would probably do the job. It is also likely that 2% lowers a bit the gelling temperature of the standard agarose, so that you can pour it not too hot on the tissue. Still, you will find it hard to pour it even at 42 °C, and it does not adhere so well to the tissue... But you are bound to be a scientist, you should try it for yourself and decide accordingly (use a trial sample, something not valuable...)!

Hello Pietro,

Thank you very much for detailed explanation! As I am the only one who does neuroscience research in our group, I was having difficulty assessing brain sample. Your answer was really helpful! Best.

Hey Pietro, really helpfull answer!

I had the same problem regarding the melting temp of the agarose.

Thanks for the help.@Pietro Pilo Boyl

Pietro Pilo Boyl

Hello again, (referring to your previous answer) Do you know any suggestible protocol, particularly including removing agarose procedure?

Thank you very much for valuable information!

Hi Yuka,

I'm sorry, I can't provide anything "published", I guess this is not the type of things described in Material and Methods... I can just tell you what we do:

To embed: we pour a thin layer of LM agarose at 37 °C in a plastic mold placed on the bench (like the ones used for paraffin embedding, or TissueTek embedding for cryosectioning... there are of different sizes), then we place the tissue on it and cover it with the rest of the agarose. When orientation of the tissue is critical and difficult, we keep the mold on a plate at 37 °C (like the ones where you dry sections cut at the microtome, but you can also use a heating block at 37 °C... if you have the ones with removable metal blocks, even better, just turn the block upside-down and you'll have an even surface at 37 °C on which to work). When you are, done just move the mold to the bench or on a cold surface.

To cut: Remove the agarose block containing the tissue from the mold by cutting the mold along the edges with a scalpel. Glue the block to the object holder of the vibratome. Trim the agarose if you have too much of it on the sides of the tissue, typically 1/2 cm is enough. Fill the chamber with ice-cold PBS and place ice all around the chamber: remember water has highest density at 4 °C, so your tissue will be hardest around this temperature and cutting will be smoother and uniform.

After the cut, pick up the slice with a Pasteur pipette where you have broken the thin side and placed the rubber bulb there, so that you use the top of the Pasteur, larger, to suck up your slice. Place the slice in a 48-well (or a 24-well, depending on the size of your tissue) and with two thin brushes remove the agarose: with one brush gently hold the slice still in the middle, with the other carefully tear off the agarose all around the tissue. It's just a matter of practice, after a while you'll be able to do this while the vibratome is cutting the next slide... Cut at slow speed to have better slices, if you go too fast the blade will push the block forward and you will cut thicker at the beginning and thinner towards the end.

Hopefully this helps.

Hi!

Pietro Pilo Boyl

Thanks for your detailed explanation.

I am using the vibratome to slice fresh tissue. The tissue I am working with is tumor(porous and not homogeneous). I embed the tissue in 4% LMP agarose and each time gel block breaks or tissue jumps out of the agarose block. My protocol is very similar to yours, but I did not fill the chamber with ice cubes. Do you recommend any substitute for agarose that adheres better to the tissue? any comments would be helpful.

Hi Dina,

indeed the 4% LMP agarose is quite rigid, you can try to reduce the %age to 2-2.5%, this should make it softer and more adhering to the tissue, also because it should compact less (decrease volume) upon gelling.

One point that might aid the cutting, use slow speed! Not more than 0.2 mm/sec, otherwise you push too much on the block you are cutting through and tear instead of cutting... That could be a reason for your breakage. Leave the vibrating blade the time to really slice your block with the horizontal movement...

Good luck,

Pietro