TITLE: Embedding and freezing fresh human tissue in OCT using isopentane
AUTHORS: Kenny Roberts, Liz Tuck

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">For many cutting-edge spatial transcriptomic analysis methods, it is essential to prepare and store tissue in a manner that preserves RNA integrity, tissue morphology, and spatial orientation.</div><div class = "text-block">This protocol describes the process of freezing fresh or fixed and cryoprotected tissues in optimal cutting temperature compound (OCT) using an isopentane-dry ice slurry. Rapid controlled freezing preserves RNA and mimises ice damage, while embedding in OCT prepares tissues for future cryosectioning.</div></div>

[STEPS]
?. [Pre-embedding and freezing (fixed frozen only)]
For tissues that are to be fixed prior to freezing, transfer tissues to 10% neutral-buffered formalin (such as a CellStor Pot) and fix at 4°C or room temperature for between 2 and 36 hours, depending upon the tissue and application.
?. [Pre-embedding and freezing (fixed frozen only)]
Fixed tissues may be cryoprotected prior to embedding and freezing. Prepare a 30% (w/v) sucrose solution using sterile deionised water. Transfer the tissue directly from formalin into 30% sucrose, and incubate at 4°C overnight. Most tissues will initially float in 30% sucrose, but sink once permeation is complete; at this point, cryoprotection may be considered complete.Cryoprotection, while desirable, is not recommended for non-fixed tissues due to the possibility of molecular degradation.
?. [Embedding]
Prepare an isopentane-dry ice slurry. Place a small metal box onto dry ice, and fill with isopentane to a depth of several centimetres. Slowly (to minimise violent boiling) add dry ice to the isopentane. When sufficient dry ice is added, the mixture will reach approximately -78°C and adding further pieces of dry ice will not induce strong bubbling (the temperature may be monitored with an appropriate thermometer).
?. [Embedding]
Label cryomoulds with both sample and orientation information; it is recommended that at least one axis, e.g. dorsoventral, anteroposterior, is noted on the cryomould. Fill cryomoulds with OCT then leave for at least several minutes; this will minimise bubbles.
?. [Embedding]
Pre-chill ~20 ml of sterile PBS (or HBSS) on ice. Fill a medium-sized disposable Petri dish (~150 mm) with ice, and place onto this a smaller dish (~30-60 mm) to be filled with the pre-chilled sterile PBS.
?. [Embedding]
Process tissues one at a time. First, rinse tissues in pre-chilled PBS on ice, as prepared above.Prior to embedding and freezing, it is recommended that tissues are examined using a dissection microscope. This will yield maximum spatial anatomical context that may be important in later sample preparation and experiments.
?. [Embedding]
If necessary, use a disposable scalpel to trim or dissect the tissue to yield multiple pieces or a specific anatomical region, or to separate tissues that were collected and shipped together.
?. [Embedding]
Following all necessary examination and dissection, use a perforated Moria spoon to pick up the tissue. Quickly and gently wick away any excess buffer using blotting filter paper. For most tissues, the sample should not come into direct contact with the paper - this may cause damage. Instead, place the tissue sample on the spoon, and either bring a corner of the filter paper to any surrounding liquid droplets, or place the perforated spoon onto filter paper and allow the liquid to seep through the holes. Take care, especially with delicate foetal tissues, not to damage the tissue sample by catching it on the filter paper.
?. [Embedding]
Using a spoon or spatula, transfer the tissue into a cryomould pre-filled with OCT. If necessary, use forceps or a small spoon to gently 'stir' the OCT to remove streaks of any residual buffer (these should be visible by eye). Then carefully manipulate the orientation of the material to suit future cryosectioning. If using fine forceps, take care not to puncture or rip the sample. Some samples will initially float, and should be gently pushed down to the bottom of the mould.It is recommended that a photo or sketch is taken of the tissue in the cryomould in its final orientation. Amend or complete any orientation markings on the cryomould prior to freezing.
?. [Freezing]
Use a pair of forceps to place the cryomould into the isopentane-dry ice slurry, keeping it level during movement to prevent loss of OCT and sample movement.Ideally, rest or hold the cryomould immersed in the top of the isopentane liquid until the OCT has started to freeze; if isopentane floods the top of the OCT early during freezing, it may promote the presence of bubbles in the block.The duration of freezing will depend upon the size of the tissue and the cryomould, but most tissues smaller than ~500 mm3 in an appropriately sized cryomould will be frozen sufficiently within 5 minutes. If the sample is slightly deeper than the mould, additional OCT may be added to cover it once the bottom of the mould has frozen, but take care not to let any isopentane flood the top of the frozen OCT prior to further OCT addition - this may cause the two pieces of frozen OCT to separate later.
?. [Freezing]
Once completely frozen, lift the cryomould out of the isopentane-dry ice slurry, and onto fresh dry ice, to allow excess isopentane to drain and evaporate. The sample may be paused on dry ice while others are processed.
?. [Freezing]
Wrap each sample, frozen in individual cryomoulds, in aluminium foil. This keeps samples clean, and separate, and minimises dehydration in the -80°C freezer. Minimise the time each sample spends away from the dry ice while wrapping to minimise temperature changes. Carefully label the outside of the foil prior to wrapping around each sample.
?. [Freezing]
Transfer samples to a pre-cooled cryobox and store at -80°C.