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

By M. A. Hayat

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The buffer is stable for several weeks at 4°C. 4%) is added to adjust the pH; at pH 5 . 5 - 6 . 0 the powder is completely dissolved. After the required pH is reached, more distilled water is added to make up 100 ml. The stock solution is stable for several weeks at 4°C. 8 g/100 ml water All solutions should be stored in bottles having ground-glass stoppers. 1 7 11 28 1. 4 ml 30 1. ) PREPARATION OF TISSUE BLOCKS Generally, the tissue blocks used for fixation should be as small as possible. If the animal needs to be anesthetized, ether or sodium pentobarbital is recom­ mended.

4) is allowed to flow through the vessel. The fixative can be dyed with methylene blue to monitor the proper flow of the fixative. The saline bath in which the section of the aorta has been immersed is replaced by 3 % glutaraldehyde. The fixative can be recovered as it flows out of the system and poured back into the container for recirculation so as to maintain the desired height of the column (h in Fig. 1) of the fixative. 34 1. C h e m i c a l F i x a t i o n Fig. 1. Diagram of the apparatus used for perfusion of the aorta.

Fixation 37 lumen of the ventricle, and the microperfusion by the fixative is started. The start of the perfusion is accompanied by the escape of blood through the opening in the atrium. 4) containing 2% polyvinylpyrrolidone (PVP). The embryo is bleached and becomes yellowish immediately. The speed of fixation can be observed by adding alcian blue or astra blue 6GLL to the fixative; all the capillaries show fixative penetration within the first few seconds. The perfusion is continued for \-2 min.

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