Basic Techniques for Transmission Electron Microscopy by M. A. Hayat

By M. A. Hayat

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Sufficient pressure (60-100 mmHg) is main­ tained to give a flow rate of 10 ml/min. A total of 2 0 - 1 0 0 ml of the fixative is perfused per fish, depending on its size. 38 1. 2 cm) is fitted. A height of 1-2 cm allows sufficient pressure. After the perfusion, the fish is covered with moist towels, and 3 0 - 6 0 min is allowed for in situ fixation. Yellowish coloration in the tissue indicates the completion of fixation. For further details, see Hinton (1975). , 1982). The fetus is delivered through a hysterotomy, and a salinefilled glove is placed over the head to avoid expansion of the lungs.

The fixed brain is placed in the fresh fixative at room temperature. Small pieces of the fixed brain are further fixed for 30 min and then postfixed with 1% O s 0 4 . , mouse) is anesthetized by intraperitoneal injection of sodium pentobarbital (30 mg/kg body weight). The animal is laparotomized and the uterus is exposed. An opening showing the yolk sac is dissected in the uterine wall under a stereomicroscope. Care should be taken not to disturb the circulation of the conceptus. The beating heart of the embryo is located, and a micropipette with a tip diameter of 2 5 - 5 0 μπι is inserted through enveloping membranes and the precardiac wall into the atrium (Fig.

As a rapid perfusion flow is desirable, the container holding the fixative is held at a 3 height of 1-2 m above the animal. Tissue cubes of 1 m m are excised from the yellow and hard tissue and fixed in the same fixative for an additional period of 1 hr. 1 Μ cacodylate buffer for 1 hr. Vapor Fixation and Staining Method 1 BEEM capsules (no. 00) are modified by cutting the lid off at the point of attachment to the capsule, leaving the polyethylene hinge attached to the lid to facilitate subsequent handling of the capsule chamber (Shepard and Mitchell, 1980) (Fig.

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