BOT/MICRO/ZOOL 5364

HEART MUSCLE PERFUSION

1.        Make up isosmolar osmium at least 2 hrs. before fixation.

For 1 g of osmic acid, dissolve in the following -

60 ml - 100 mM sodium cacodylate pH 7.4

12 ml - 53.2 g/1 sodium chloride + 4.42 g/1 KCl

12 ml - 2.06 g/1 CaCl₂ + 1.138 g/1 MgCl₂

36 ml - H₂0

Place on shaker and shake until dissolved.  Can be made up the day before and kept in refrigerator overnight.

2.        Make up Ringers solution

For 1 liter                                            pH to 7.40!

100 ml - 4.74 g/1 Tris maleate + 2.34 g/1 N acetylglycine

100 ml - 89.6 g/1 NaCl + 4.42 g/l KCl

100 ml - 2.06 g/l Cacl₂ * 2H₂0 + 1.138 g/l MgCl₂ * 6H₂0

3.        Make sure to have 1% aqueous uranyl acetate available.

4.        One hr. before fixation heat water bath of perfusion apparatus to 37°C.  Place Ringers containing 3 ml of heparin solu. in one side.

5.        About 30 minutes before fixation inject rats with 1 ml of approx.

1:4 heparin-water mixture.

6.        Set up apparatus with Ringers and osmium so there is no air in the system.

7.        Start Perfusing with Ringers when hear is on cannula.  Switch to osmium when the coronary effluent runs clear.  Fix for about 7 min.

8.        Take heart down and keep wet.  Separate right and left ventricles.  With razor blade, cut tissue into small pieces using, fresh part of blade each time.

9.        Wash 3X in Ringers 5 minutes each.

10.      Place pieces in 1% uranyl acetate overnight in the refrigerator.

11.      Place tissue in 25% EtOH - slosh and remove.

 50% " - " "

 70% " - " "

 95% " - " "

100% " - " "

100% " - " "

12.      Continue as follows -

1:2 of plastic: 100% EtOH - Shake gently 15 min.

2:1 of plastic:   "  "       "    "      "  "

100% plastic -- Shake gently 15 min.

Put chunk in BEEM capsule with plastic in them.

Put into oven.