Hi,
Perhaps I've been searching wrong, but I've been unable to find a satisfactory answer for a good extracellular cryoprotectant in high pressure freezing. I know there are many options but each seems to have drawbacks.
Examples: Hexadecene is a good cryoprotectant, but it supposedly inhibits freeze substitution. Alternatively, 20% BSA doesn't inhibit FS, but can create a brittle black disc one could mistake for their sample.
Does anyone have any pointers on this? I'm working with 2mm tissue punches in 300um deep brass wells. Ideally I'd like to be able to use McDonald's SQFS method. The last time I used the SQFS method it was very close in preservation to the typical 5-day FS protocol.
Thanks in advance!
Hi Colin,
I'm always using 20% BSA and it gives satisfactory results. It turns brown after embedding, but usually the sample is darker and I never had problems finding it.
Regards,
Andreas
Hi Colin,
I'm always using 20% BSA and it gives satisfactory results. It turns brown after embedding, but usually the sample is darker and I never had problems finding it.
Regards,
Andreas
Hi Collin,
You can scratch the hexadecene off the sample after freezing using a cold needle, it is generally more brittle than a well frozen sample.
20% dextran is another option but only if you don't add water to the freeze-substitution medium (this prevents adequate removal of water from the dextran).
2-methyl-pentane is another interesting filler, since it is still liquid at -140C
and doesn't interfere with freeze-substitution. Loading the samples is a bit more difficult since it evaporates rather fast. (filling the carriers while they are submerged in the 2-methyl-pentane is the easiest).
I think the biggest improvement you can get in terms of preservation of your sample is by using less deep sample carriers. The upper thickness limit of HPF is around 200um, with a cryoprotectant as filler.
Regards,
Rob
Unfortunately, a more shallow carrier is not an option.
If I recall right, the theoretical limit for HPF is about 600um. I know that good freezing at that thickness is rarely the case. But from what I'm getting at 300um, it seems that adjusting the placement of tissue (I'm only interested in a particular cell layer) and the cryoprotectant should result in better freezing.
I'll look into methylpentane!
Hi Colin, did you actually experience problems with FS when using hexadecene? I've never had an issue with Hexadecene inhibiting FS, not even for SQFS.
For thicker samples, besides what has already been mentioned above, I like to use yeast paste. Mix some old bakers yeast into the buffer of your choice to make a thick paste (I additionally inactivate the paste in a microwave, otherwise it might form bubbles). Transfer the paste on filter paper wetted with buffer. Apply the paste with a wetted soft hair brush.
Good luck,
Martin
Dear all,
We also use 20% BSA in the sample's buffer. A tip for preparation :
- 850ul of the given buffer in a 2 ml eppendorf tube
- Make sure that there is no drop of buffer on the side of the tube
- Weight 0.2g of BSA
- Add it to the tube as a layer on top of the buffer
- Centrifuge at 10000rpm for 10'
Done !
Nicolas
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