NMOpticsDude, on Apr 11 2009, 04:51 PM, said:
I am an optical scientist who is clueless about using human tissue in the Lab. Note that we are a small business, NOT a university.
We are writing a grant proposal for NIH to optimize some optical parameters for some commonly used instrumentation (like OCT, etc.). To confirm our physics, we need to test in real human tissues which probably will include the following :
Retina or Cornea
Can you enlighten me about
1. Proper storage to preserve tissue quality
2. Proper safety procedures
4. Sterilization (do we even need it at all to meet certain guidelines?)
Are there companies that we can pay to do most of this for us? i.e. deliver the samples, then pick them up when we are finished and properly dispose of them, etc. Then all we would need to do would be to properly store the tissue samples until we are finished using them (I assume a normal refrigerator would suffice).
All comments, suggestions, and advice will be appreciated,
Ok, so lets try to put it in short and let you come back with specific questions.
When you collect tissue you may want to use it for quantitative(or semi) analysis or for histological analysis.
This means you either want to homogenate (make a milkshake) or cut (like cold meat) the tissue.
1)If you want to homogenate it simply subtract it, place it in a tubes and freeze it till homogenization.
If you can, instead of freezing till homogenization, homogenate it after subtraction.
If you pretend to work with RNA use DEPC water, clean everything with RNAse free, change gloves...
2)After homogenization centrifuge to pellet insoluble material.
3)Then aliquot in ice and store at -20 (-80 is usually preferred).
For histological analysis:
1)After tissue retrieval insert it in paraformaldehyde 4% (either 1h ar RT or overnight at 4º).
2)Wash excess paraformaldehyde in PBS 24h 4º (or more).
3)include in parafin and cut
1)After tissue retrieval place in a thin plastic-vynil small container cover the tissue with OCT and freeze, cover with aluminium foil and store at -20º/-80º.
2)Cut the block
3)Fix in cold acetone
*It is recommended to freeze samples as soon as possible to stop protease-rnase activity. For the same reason when not frozen it is recommended to keep samples on ice.
*It is recommended to freeze fast to avoid crystal formation inside cells that breaks them and impairs tissue histological recognition.
*To freeze fast (snap-freeze) you can you use liquid nitrogen (an old stardard) or an slurry of dry ice-isopentane(freezes faster as it doesn't form a vapor layer around the sample like liquid nitrogen, (air has very low thermal conductivity)) .
*Never hold dry-ice in a closed container like a bottle it expands till explode, in fact it is a kind of bomb.
*Some enzymes still active at -20º, and as salt content increases freezing point decreases, that's why -80º is usually preferred. The first point doesn't make much sense to me, as at -20º the tissue is frozen so the enzymes can't displace. The second point has to be more with manipulation as, if freezing point is at -10º, litle handing may unfreeze the samples.
*Unfreezing leads to tissue impairment, enzyme activity and protein denaturation that's why stuff is aliquoted, to avoid repeated freeze- thwa cycles.
*Paraformaldehyde leads to better morphology but may impair probe recognition.
My best recommendation for cutting skin and fat is to remove as much OCT as posible while cutting. These stuff physical properties are to different from OCT. In fact, this is my best recommendation for every tissue, but specially for those that give trouble.
I have a friend that works with blood, of course you need EDTA to avoid coagulation and it is advised to separate phases by centrifugation, especially he recommends to remove eritrocite.
To work with retina it is adviced to remove cristalline to help buffers enter the eye and contact the retina. For homogenates it is common to remove the epithelium pigmentarium as it is sticky and protein rich.
Use globes during cutting.
Disposal depends on your local recycling company, they should provide you with special containers and collect them from time to time or when you call 'em.
To avoid fungus growing you can use Thimerosal or sodium azide (avoid the later if HRP is to be used).
To sterilize you can use heat, UV, fire or simply wash it