Currently I am doing research into muscle composition in mice. The focus is on two muscles with a diameter of around a millimeter, which I am sectioning with a Cryostat. The muscles were on tension snap-frozen in liquid nitrogen. Therefore, the tissue still has to be embedded in OCT. This raises the risk of thawing, for which I currently use the method to dip the muscle in OCT and directly freeze the complete clot in -80C isopentane.
The problem is; my tweezer will be frozen to my sample/the OCT. Getting the sample off the tweezer has the risk of breaking the muscle. Can someone share good practices for getting the specimen of my tweezers?
What temperature is your tissue currently stored at -80 degrees or - 20 degrees?Are your samples in a cryo vial or wrapped up in tin foil or just loose in a tube of some descrip? Are your samples fixed (4% PFA for example)Are your samples Cryopreserved ? were they submerged in 30% sucrose post fixation?
Normally cryo sectioning of skeletal muscle is in a cryostat with a chamber temperature of -20 however the cutting temperature is nearer -18 degrees. Fixed tissue can withstand a degree of 'abuse' that unfixed cannot. Cryoprotected tissue will appear less freezer damaged that non protected especially if stored at -80 degrees. Normally fixed tissue is perfectly safe and more robust at minus 20 and NOT minus 80.
Noting all the above questions: tissue unfixed without cryopreservation stored at minus 80 will present the greatest challenge. Tissue will 'chit' producing freeze fracture artefacts .sections will appear lined.Staining will be uneven with areas where almost no satin is picked up.
Unfixed uncryoprotected stored at -20: transfer direct to the cryostat. Have an ample supply of dry ice at the ready for immediate local temperature regulation. You don't want to adjust the whole cabinet temp just because your block is slow to freeze. Stick forceps in dry ice THEN pick up tissue and place on a blob of wet/fresh/unset OCT which is on the chuck (the platform your block sits on). Transfer wet OCT with tissue into SMALL container of DRY ICE IN CRYO cabinet. (empty pipette tip box). You don't want to wave samples about in air temp. Try to maintain equivalent temp throughout the whole process. Orientate before OCT sets. Place chuck with tissue in OCT carefully to the back of the cryostat and process the next sample. While you're breathing into the cryostat the temp will increase BUT dry ice helps to retain the setting.
Keep your paintbrush/forceps/blade in dry ice at all times so further manipulation of tissue or block doesn't alter temp of tissue.
Fixed & cryopreserved stored at -20 should be No problem - 'defrost' sample in blob of OCT in cryostat (going from minus 20 to approx minus 18 degrees) and section as per
Fat sticks & warms up at a different rate so use your chilled blade to take this slices of offending from block. Use a spike of dry ice to chill the freshly cut region and use a Real hair paintbrush to brush any OCT/fat away from block (Don't use nylon which 'pings' when frozen and can stab your tissue).
Check (light microscope) quality/orientation of sections after about 5th section. make necessary adjustments. Enjoy !
Dear Caroline and Hussein,
Thank you for your replies! The samples are already stored in -80 degrees, just loose in a tube and not cryopreserved. The reason for this is that we wanted to section the muscles 'on tension'. Therefore I will not be able to use the procedures both of you described.
Still; thank you for your accurate descriptions, I will bear them in mind in future!
Frans
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