|From:||"George Cole" |
1. ALL of the tissues sent will be frozen and sectioned. There is no way to look at 100% of the tissues---but we can represent every portion of the tissues. With normally sized biopsies, pieces to be cut may be just slightly narrower than the cover glass or a group of several narrow pieces just narrower than the cover glass can be frozen side by side. Pieces are best processed if they are no more than half a cm thick and long. Huge biopsies a cubic inch and larger occasionally arrive. Pieces a cm long and almost a cm thick have been frozen all right, with occasional minor ice crystals in the center. As much of each segment of the tissue should be represented as well as possible in the sections.
2. OCT is the mounting medium of choice. It is compatible with all of the histochemistry. Only a thin coating of OCT is put in the bottom of the mold to hold the tissue in the mold during freezing.
3. Those thin walled plastic molds work just fine---they have a name, but I never knew it--- They come in at least two different sizes. About a ¾ inch square cavity, and the other a rectangle of about an inch or so by half an inch
4. A thin coating of OCT is put on the bottom of the mold to act as glue, so the tissue won’t pop out during freezing.
5. A thin walled aluminum cup----2.75 to 3 inches diameter at the brim and slightly narrower at the bottom (mine was meant to be a gravy mixer) is filled with a cup or more of isopentane and lowered into the liquid nitrogen. The cup is best held with long forceps. When the fizzing stops for the second time---there is usually a secondary roiling of the isopentane----a SMALL bit of liquid nitrogen is admitted into the cup ( No need my telling you a small bit---use more---you won’t want to do it twice---it boils all over the place! ) and stirred. I used a butter knife with thick wrapping of paper towels around the handle---it gets COLD. This is done until you have. a thick slurry. The muscle in the mold is quickly put into the slurry and dunked to a slow count of 10 to 20. The mold is lifted out of the slurry, shaken twice quickly, to shake off the clinging isopentane, and put in the liquid nitrogen for 10- to 20 slow dunks. Then the mold is put directly into the cryostat.
6. The tissues are removed from the mold inside the cryostat. The frozen OCT with the muscle in it is placed on a specimen holder for your cryostat. It is then bolstered about with OCT ---the bolstering results in a neat rectangle wider across than up and down. All of the edges are trimmed to be straight and clean---a straight edged, square cornered rectangle which is cut with the broad bottom to the knife. You will see that with edges straight and neat, you will have happier time cutting sections. Go watch the residents sometimes, cutting frozens with the bare tissue turned any which way, splitting, turning, getting cussed.
7. Sections are put onto 2 by 3 inch glass slides. Using cover glass is a volunteering for misery. I can’t imagine how that got started. You can handle and store the glass slides easier and 178.56 times safer than when on cover glass.
8. Reagents are mixed to FILL the coplin jars, completely covering the tissues on the slides. Stingy volumes of histochemical preparations is inherited thru the genes from Scrooge!!
9. Sigma sells a buffer called 221 that you will see used in the ATP-ase and the other preparations in the packet.
. It does not require a controlled substance license to purchase and it beats the socks off the sodium pentobarbital in all functions. =A0
All of this is better covered in the video.
Now then, what’s going to happen? What will win out there in your labs----habit or function??