Hi Eve,

I do not work with skin. However, in our lab we work with rodent brains and we actually do practically the same process like you to freeze them.

Sometimes we keep them for a long time (6 months could be) at -80ºC without OCT. We freeze them in isopentane at -30 - (-40) ºC and then we put the brains in the aluminum foil without OCT. However, if you embed it with OCT you need to go as fast as you can to avoid melting it.

I hope you find it useful,

Jordi.

Thanks both. Jordi - so are you saying you freeze the brains without embedding them in anything? Do you then embed the frozen tissue in OCT for sectioning?

Hi Eve,

Yes, we freeze and keep them at -80ºC. Just before cutting it, we leave the brain 30 minutes in the cryostat. Afterwards, we embed the tissue with OCT and we "play" with dry ice to keep it frozen and not melting. When the block is totally solid we cut it.

Jordi.

@Jordi:

honestly I am allowed to ask whether in your Re #[002] there was an erroneous setting/writing a numeral, namely of "-(minus) 30°C (-40)°C" or you definitely do so.... since I've never heard/read about freezing in isopentane at such a low (high.... it is relative from which end you'll observe this (:-)) ) temperature, leaving / storing then your specimens at -(minus) 80°C (wrapped in Al-foil to prevent desiccation) until sectioning. There is the possibility that you cooled Isopentane by means of dry ice (but this also will lower the temperature only to ~minus -78°C). If it is a typo (-30°(40)° instead of say-150 to - 160° degr. C), please correct that error by editing! Thank you!

Isopentane (2-methyl-butane) usually is used in freezing delicate biopsy specimens (e.g. muscle). The liquid isopentane-contained in a separate - sometimes conical or hemispherical receptacle - is cooled down by circumfluent LN2 (liquid nitrogen) until the fluid at approx. minus 150°-155°C begins to get / become solid / crystallize (Freezing point of Isopentane= minus -160°C) appearance like a 'slurry'). That is the "golden" time point where the nearly solid isopentane has high cooling capacity and provides also high cooling rate for small(er) specimens, which are (eventually somehow precooled in the nitrogen fog / vapour from the LN2-Dewar) thrown in the receptaculum. The limiting factor is the "Leidenfrost-phenomenon" [thermic isolation due to vigorous LN2 vapor generation) which can be reduced if the surface of the specimen does not contain much adhering moisture.

Sources:

e.g. (not exhaustive):

http://www.uab.edu/research/administration/offices/ARP/ComparativePathology/Pathology/Histopathology/TissueSubmission/Pages/Freezing-Tissues-for-Cryosectioning.aspx

https://www.google.com/url?

https://www.researchgate.net/post/Is_there_a_way_to_freeze_tissues_for_cryosectionning_without_sucrose_or_OCT

YouTube: " Snap freezing muscle in Isopentane/Liquid nitrogen double bath"

(This video IMHO shows unfortunately a 'risky precooling' of isopentane in a beaker-glass? put into a larger plastic bowl containing LN2 already. Only use plastic beakers and let equilibrate to prevent generation of too much LN2-vapour. Personal Protective clothing / equipment with at least Safety goggles and suited gloves to prevent combustion IMHO are mandatory):

sa=t&rct=j&q=&esrc=s&source=web&cd=1&cad=rja&uact=8&ved=0ahUKEwidvp7hqbfcAhXJZ1AKHT4sD1kQwqsBCDEwAA&url=https%3A%2F%2Fwww.youtube.com%2Fwatch%3Fv%3DL09teZ9mm_Y&usg=AOvVaw1-A4hmvHzfcTZQbbKzPtjP

Flash Freezing and Cryosectioning E12.5 Mouse Brain:

(Institutional or private Subscription necessary)

https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=2&cad=rja&uact=8&ved=0ahUKEwidvp7hqbfcAhXJZ1AKHT4sD1kQwqsBCDQwAQ&url=https%3A%2F%2Fwww.jove.com%2Fvideo%2F198%2Fflash-freezing-and-cryosectioning-e125-mouse-brain&usg=AOvVaw2qKeANe4F_D4cORBcgKAYn

from Archives Histonet, Oct.2003: http://www.histosearch.com/histonet/Oct03/Whydryiceisopentaneslurry.html

(NB: you might check for ~6 (six) and (at least 2=two) Message (cf. link at bottom of page) to find the original question and other replies to the thread with the title: " Why dry ice isopentane slurry for snap freezing?" or "Lovely set up for snap freezing". Also read the previous answers contained in each 'new' post)

further: at:

http://www.histosearch.com/histonet/Oct03/Histonetfreezinginisopent.html and

http://www.histosearch.com/histonet/Oct03/Histonetfreezinginisopent.html

http://www.histosearch.com/histonet/Oct03/RE.Histonetfreezinginisop.html

http://www.histosearch.com/histonet/Oct03/RE.HistonetfreezinginisopA.html

additional information (ads of companies dealing with freezing devices)

Hi Wolfgang,

In our lab we use this method: freezing with isopentane between -30 and -40 (using dry ice to cool it) and then dry ice just before storing it at -80ºC. We have never had any problem and our tissue keeps a very good morphology.

However, I am totally aware that many labs do it just with dry ice not usingisopentane and they obtain the same quality.

Thanks for your answer. It is easily observable your expertise in the field. I truly believe that your reply is absolutely complete and formidable.

Jordi.

Dear Jodi,

Thank you so much for your kind and fast reply.

This your method seems interesting because you do not cool down isopentane at a temperature beyond the recrystallisation temperature (which 'usually is reported to be' minus 80°C; Dry Ice exerts a temperature of minus 76 till 78 degrees C, and this only if you incubate the isopentane as long as necessary to reach thoe minus 76 °C). How do you measure the -30 / -40 °C (since I presume you have adopted and follow kind of standardized protocol) you may be certain to start the freezing procedure?

I haven't seen the results you obtain after such freezing, but in terms of preservation of the overall morphology / appearance of your brain specimens as appearing in stained "histological cryo sections" you obviously are satisfied and do not see freezing artifacts, do you?

Concerning your statement ".... do it just with dry ice not using isopentane and they obtain the same quality. " I greatly should appreciate a rationale for your using isopentane at a rather higher temperature than usually needed for rapid and (most desired) effect of freezing without formation of ice crystals...

Therefore it would be nice to see some images of your section preps....

(doubting "Thomas")...

And last but not least: thanking you 'for the flowers': "I truly believe that your reply is absolutely complete and formidable." but: hopefully that doesn't imply I should shut up...(:-))

Yes, I was confused about embedding already frozen tissue in OCT.

But generally, most people think it would be best to freeze down this tissue and leave it in the -80, rather than leave it in sucrose at 4 degrees?

Thanks everybody!

@Wolfgang:

You are totally certain. It is a standarized protocol that we have been using in our lab in many experiments (we perform a lot of in vivo ones). We basically keep the isopentane in dry ice but with some pellets (not a lot) and then with a thermometer we can measure whether it is in that temperature (going to -80 we could not know with this method due to the thermometer). Ostensibly, we have not seen any artifact, thus, we have not considered to change the method. And, obviously, the rationale is the empirism besides the technique and its results.

To cut a long story short, I was not expecting you to do so. I was just pointing out the unarguably truth (:-)).

@Matthew:

If you do not go very fast, some cells can thaw due to the temperature shock. We are interested in a inner part of the brain and we have not seen any thawing process. However, in the periphery (cortex) some labmates and me have seen it. The most experienced ones can avoid it going fast and well.

J.