The effect of fixation on the autoradiographic grain count

Abstract

AMONGST the most popular fixatives of animal tissues for light microscope autoradiography are formalin, ethyl alcohol, ethyl alcohol mixtures (e.g. those of Belanger and Carnoy), and glacial acetic acid mixtures (for example, Bouin’s and its modifications). It is reasonable to expect that the fixative might be important in subsequent autoradiographic studies, and KOPRIWA and LEBLOND (1962) have obtained significantly different counts with different fixatives of pieces of liver from a 3H-cytidine injected mouse. This report outlines a satisfactory technique for light microscopeautoradiography of developing dental tissues and compares the effect of several fixatives on the autoradiographic grain counts from ameloblasts labelled with tritiated proline. Fourteen-day old albino rats (Wistar strain) were injected with 8 PC per g of body weight of jH-proline (Radiochemical Centre, Amersham, England; spec. activ 253 mc/mM). The animals were killed by decapitation at intervals of 20 min, 60 min, and 90 min, after the injection. Each head was quicklydissectedinto fourjawquadrants, which were each placed into one of four fixatives : buffered neutral formalin, Hollande modification of Bouin’s fluid (JONES, 1966), 3 per cent glutaraldehyde in sodium cacodylate buffer, and glutaraldehyde with osmium tetroxide post-Gxation. The first three were processed for light microscopy and the fourth was processed and kept for subsequent electron microscopy. The tissues for light microscopy were fixed for 3 days at room temperature, decalcified in 10 per cent EDTA, washed in several changes of distilled water and embedded in paraffin. Sections 6 p thick were placed on gelatinized slides and prestained with Mayers haematoxylin and eosin. The liquid emulsion technique used for autoradiography came mainly from the methods reported by KOPRIWA and LEBLOND (1962), KOPRIWA (1966) and ROGERS (1967). Ilford K2 emulsion was diluted with an equal volume of 0 5 per cent glycerol in distilled water and all slides were coated in a uniform manner by means of a dipping machine (KOPRIWA, 1966); the time of withdrawalof each slide from the emulsion was 42 sec. The back of each slide was wiped clean and the slide was then placed to cool, face downwards, upon a chilled glass slab. The slides were stacked in Clay-Adams light-tight boxes which were flushed out with COz before sealing, and the sealed boxes of slides were stored at 4°C in a lead-lined container. Each box contained sections from all three fixatives. All slides were developed after 8 days in the following developer: 2 ~2 g sodium sulphite (anhydrous) plus 1 *O g amidol, made up to 620 ml with distilled water and