• Dissection instruments and board
• Perfusion fluid (see Protocol 6)
• Length of fine Portex plastic tubing (size 23G) (Fischer Scientific)
1 Fill the 20 ml syringe with heparinized PBS and the 50 ml syringe with periodate-lysine paraformaldehyde solution (see Protocol 6).
2 Stretch the plastic tubing between fingers until quite thin.
3 Cut obliquely across the tubing at the thin section and attach the butterfly needle to the other end.
4 Connect the 20 ml syringe to the butterfly needle and test to ensure good flow.
Protocol 7 continued
5 After sacrificing the animal, secure the animal on the dissection board and spray with 70% alcohol.
6 Thread the plastic tubing under the animal's left hind leg, for stability, and in a curve round to the heart area,
7 Make an incision to expose the abdominal organs and one down each side of the ribs to expose the heart.3
8 Introduce the obliquely cut end of the plastic tubing into the left ventricle of the heart.b
9 Make an incision in the inferior vena cava, just above the right kidney, to allow the fluid to drain.
10 Run the heparinized saline through the body at an approx. rate of 2 ml/min.c
11 When the body fluid appears to be clear upon exit from the kidney area, change the syringe to run the fixative through the animal at approx. 2 ml/min.d
12 Continue to perfuse the whole 50 ml of fixative (perfusion fluid) to ensure good fixation.
13 At the completion of perfusion, dissect the animal.
14 Post-fix the organs in the same fixative for about 4-6 h.
15 Place the organs in 20% sucrose in 0.1 M phosphate buffer overnight.
16 Block the tissue (see Protocol 5, steps 4-8), taking care to ensure all moisture is absorbed on a tissue before embedding.
a Leaving the uppermost portion of the ribs gives support to the heart against the pressure of the fluid.
bDo not use gloves as this will cause the tubing to slip out of the heart.
cIt is critical not to huny with this procedure as forcing the fluid through too quickly will irretrievably damage the organs and cells. If the tubing becomes detached from the heart during the perfusion procedure it is not easy to reattach without losing fluid from the old incisions. Thus, fixation will be incomplete and the procedure should be abandoned. Care should also be taken so the line is not pushed through the heart and out the other side!
d Shortly after starting to run the fixative through the animal may be seen to 'twitch'; this an indication of the fixative reaching all areas.
Tissue blocks are cut on a cryostat; usually at a thickness of 5-10 ^m. Depending on the density and hardness of the material, some tissues may require a slight change of temperature in the cryostat chamber to cut satisfactorily. For example, bone may require cutting at a lower temperature than liver. The temperature required is resolved by trial and error, however, as a guide, a recommended temperature for the ayostat chamber is -20 °C and that of the chuck, - 13CC to -18°C. The sections are collected onto slides and may be stored at -20'"C. When block cutting is finished, a small amount of Tissue-Tek should be used to cover the exposed surface to prevent 'freezer-burn' before returning to the freezer. Some thawing is inevitable when the Tissue-Tek is placed on the exposed surface after cutting, this results in some loss of antigen so if re-cutting, cut well into the block before collecting sections.
Once frozen, slides should not be allowed to thaw until required for use. It is safest to assume that freeze/thawing slides will significantly reduce antigen detection, so when collecting some slides for use return the rest to the freezer as soon as possible.
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