How long can tissue stay in 30% sucrose?
3. Place tissues in 15% sucrose (Cat # S5-3, Fisher Scientific) in PBS until tissue sinks (6-12 hrs) and then 30% sucrose in PBS for overnight or until tissue sinks. Best if the tissues are gently nutated, taking care to avoid contact with bubbles and the air surface interface.
Why is sucrose a good cryoprotectant?
Sucrose is included in the disaccharide group. Sucrose is expected to function as extracellular cryoprotectant to protect spermatozoa cell membranes from the effects of cold shock due to storage of spermatozoa at low temperatures and as an energy source for the metabolism of spermatozoa during storage.
How do you make a Cryosection?
METHOD
- Freeze a fresh, unfixed tissue sample, up to 2.0 cm in diameter, in OCT in a suitable tissue mold.
- Cut sections 5-15 μm thick in the cryostat at −20°C.
- Within 1 min of cutting a tissue section, transfer the section to a room temperature microscope slide by touching the slide to the tissue.
How do you use an OCT compound?
Put 2 drops of OCT into a plastic cryomolds. Place tissue on top in correct orientation for cutting. Carefully pour OCT on top of tissue, being careful to avoid bubbles until none of the tissue remains exposed. Place the mold on top of the aluminum plate on the dry ice for rapid freezing.
How long does it take for brains to sink in sucrose?
about 48 hrs
In conclusion, in our experience over-fixation was never an issue, so I would wash well the brain in PBS, maybe male it O/N at 4 C, and then transfer to the sucrose solution… it will take about 48 hrs to sink.
How long does it take the brain to sink in sucrose?
For adult mouse brains, it takes at least 3 days for dehydration to complete – i.e. the brain sinks to the bottom of the tube in 30% sucrose.
What does sucrose do to tissue?
We learn that sucrose will decrease the cell deformation. I think it will also limit the swelling of your tissue during the fixation processes. An old article (Bahr 1957) told that the deformation was inversely proportional to the sucrose concentration.
How do you freeze fresh tissue?
Rapidly wrap all samples in pre-cooled, labeled foil, and place in a pre-cooled plastic bag, in a freezer box. Store at -80°C. temperature for approximately 30 minutes. the tissues have been freshly frozen or pre-fixed with subsequent cryoprotection.
Does October Fix tissue?
Optimal cutting temperature compound (OCT compound) is used to embed tissue samples prior to frozen sectioning on a microtome-cryostat.
When to use sucrose before or after cryopreservation?
sucrose solutions above 10% are hypertonic and will cause water to flow out of cells and tissue shrinkage if tissues are not fully fixed. A short period of fixation prior to cryopreservation with sucrose can be used IF: 1)
Why is sucrose concentration chosen at 30%?
Normally tissue is fixed in 4% PFA (or animal perfused with 4% PFA before taking tissue from animal), than the tissue passed thru 15% sucrose and than in 30% sucrose before freezing in OCT blocks. Why sucrose concentration is chosen at 30%?
Why do we soak tissues in 30% sucrose before embedding in Oct media?
Why we soak tissues in 30% sucrose prior to embedding in OCT media? Normally tissue is fixed in 4% PFA (or animal perfused with 4% PFA before taking tissue from animal), than the tissue passed thru 15% sucrose and than in 30% sucrose before freezing in OCT blocks.
Why is sucrose used to prepare frozen tissue?
Tissue preparation and cryopreservation with sucrose — for frozen tissue sections. The purpose of cryopreserving tissues is to help prevent ice crystal formation in tissues when water freezes and expands.