Snap-frozen Research Articles

Direct Measurements of Intracellular Elemental Composition Utilizing a New Approach to Freezing In Vivo

Many biological experiments can be successfully carried out on tissues isolated under well-defined experimental conditions, but for some physiological or pharmacological studies, knowledge of the in vivo elemental composition and/or structure is essential. We have developed a method for rapid freezing of organs in situ, with a new spring-loaded, hand held, clamping device that holds two apposing melting Freon popsicles (Fig. 5). The melting Freon surfaces that contact the tissue are at the melting point of Freon 22, and these mold to the unevenness of the tissue surface. Rapidly frozen samples of liver, pancreas or mesentery were obtained by placing an anesthetized rat in a cloth sling over an environmental chamber kept at 37°C, 100% humidity (Fig. 5). A small incision is made in the abdomen, and the abdominal wall is manipulated until a lobe of liver protrudes through the incision, while taking care not to touch the surface of the organ. Immediately, the Freon clamper is activated to snap freeze the tissue.

Functional capillary density in skeletal muscle during vasodilation induced by isoprenaline and muscular exercise

Functional capillary density in skeletal muscle was studied in an isolated, autoperfused preparation of the abdominal muscles of the rat during different forms of vasodilation. The macromolecule hydroxyethyl starch (MW 450,000), labeled with the fluorochrome lissamine-rhodamine B 200, was intravenously injected. When a certain volume of blood had passed the muscle the tissue was fixed by snap freezing. In histological sections those capillaries which had been perfused by the dye could be visualized in the fluorescence microscope. Increase in total muscular blood flow, measured by arterial drop counting, was induced by intraarterial infusion of isoprenaline and by muscular work (control 30.0 ± 2.6, isoprenaline 48.2 ± 4.4, postcontraction hyperemia 44.2 ± 7.4 ml/min × 100 g). During hyperemia the functional capillary density (stained capillaries per muscle fiber) was affected in a different way: 0.81 ± 0.02 control, 0.71 ± 0.03 isoprenaline, and 0.93 ± 0.04 postcontraction hyperemia. The data support the view that a rise in total blood flow is not necessarily associated with an increase in functional capillary density.

Effect of snap freezing, dithiothreitol and storage on estimations of estrogen receptor sites

Estrogen receptor assays were performed on human myometrium in order to determine optimal conditions for certain steps of the assay. The findings indicate that: 1. (1) tissue should be snap-frozen in liquid nitrogen prior to assay, 2. (2) dithiothreitol does not increase measurable binding site numbers, 3. (3) estrogen receptors in the cytosol fraction are unstable and should not be stored in excess of 1 day, 4. (4) if storage prior to receptor assay is unavoidable, tissue should be stored at − 20°C or − 70°C for no longer than 1 week.

Disc electrophoresis of the haemolymph proteins of some portunid crabs (decapoda: portunidae)—I. Effects of storage

Abstract 1. The electrophoretic patterns of haemolymph proteins from 12 species of portunid crabs are described. 2. A comparison of haemocyanin mobilities from stored and fresh haemolymph indicated that mobility may be decreased after snap freezing and then storing at − 10°C. 3. Similar haemocyanin mobilities were recorded from 2 gel lengths and from both sexes. 4. Frozen haemolymph produced smaller clots associated with a higher concentration of serum haemocyanin than did fresh haemolymph.