|
|
|
|
Mounting tissue Mount the tissue between glass microscope slides. There are 2 good mounting protocols: a) In 80% glycerine + 20mM Na carbonate, pH 9.5. b) Place your tissue for a short time (10 minutes or so) in distilled water to wash out the salts and Triton (they can precipitate in alcohol, makes a mess), then dehydrate very slowly through an ethanol series of 10-15% steps or so, the amount of time dependent on the thickness of your tissue, ~10 minutes per step for our STGs. This takes a long time, but the results are worth it. If dehydrated too rapidly, the preparation (cells) will take a puckered appearance. Finish with a couple of washes in 100% ethanol, and mount in methyl salycylate, The mounting solution is pure methyl salycylate. Sigma's is fine, but there are other sources that seem to do just as well. Be careful with this stuff though. It is not really too toxic (it is just spearmint after all), but you can build up an allergy to it. Many common foods have spearmint in them, which you will then have to avoid. Use both gloves and a fume hood, if possible. In addition to thickness, another determining factor with crustacean ganglia is the permeability of the sheath. If you desheath, 5 minutes should be long enough, but I would say 8 to 10 to be safe. At least 8 or so minutes if you do not desheath. If you want to try 5 minutes, the symptoms of too-short washes are roughly the same as for increasing the alcohol concentration too rapidly: the cells look like they are wrinkled and puckered. Also, since salycylates are not miscible with water, sometimes you will get a milky appearance in the ganglia-basically like trying to mix oil with water. This milky thing is reversible by the way, just stick it back in 100% ethanol. Also, you can wash out the salycylate by reversing your ethanol steps, and subsequent immunolabelling or intensification steps on re-hydrated ganglia are possible. Keep your eye on your ganglia when you transfer from 100% ethanol to salycylate. If your tissues have some pigment, you may still be able to see them, but if they are like our unpigmented crayfish ganglia, they will become completely invisible. Place them in salycylate on the slides, saves having to feel around for them. Another thing to keep in mind: since this stuff does not harden, you have to be very careful about getting it on objective lenses. It can remove some types of coating. Make sure you wick out as much as possible from under the cover slip. Cover slips can be partially sealed around the edges using Permount or Entellan or any of the other organic based mounting media, but the salycylate will eventually melt through these, so its not permanent. Water bases mounting media do not work, they will be repelled from the salycylate and not seal at all. Store in the freezer if it will not harm your tissues or labels, this stuff will not freeze, but it will eventually evaporate off if kept in the fridge or at room temp.
|
|
|
