Joó, Ferenc

Rakonczay, Zoltán

English

Joó Ferenc

Rakonczay Zoltán

University of Szeged

Acta Biologica Szeged. 22 (1—4), pp. 83—85 (1976) A SIMPLE SAMPLING TECHNIQUE FOR OBTAINING REPRESENTATIVE ELECTRON MICROGRAPHS FROM BRAIN FRACTIONS F. J o 6 a n d Z. RAKONCZAY Institutes of Biophysics and Biochemistry. Biological Research Center. Szeged. Hungary (Received September 3, 1975) Abstract Subcellular fractions of brain tissue were placed on and adhered to the surface of millipore filters in a commercially available holder. Subcellular particles forming a thin layer on the filters in an even distribution could be readily processed for electron microscopic evaluation. It is expected of an electron microscopic study that it will assess with accuracy not only the distribution of particles derived from subcellular fractions, but also the proportions of various components being present in the original fraction. However, because of certain inherent difficulties such as the different sedimentation coefficient of particles and the extremely small volume of a thin section, it is hard to obtain representative electron micrographs by using conventional electron microscopic processing from small pellets of the brain fractions. We report here that, after collecting subcellular particles in an easy way on the surface of millipore filters, a small but still representative volume of brain fractions can be readily processed for electron microscopic investigation. Materials and Methods Subcellular fractions f rom rat brain were prepared according to the procedure of Jo6 and KAR-N U S H I N A (1973), and elaborated for the isolation of brain capillaries. After density gradient centrifuga-lion, four fractions (from A to D) were separately collected. D fraction, the capillary-rich pellet, was suspended in 2.0 ml of 0.25 M sucrose. Then 100 nl of each fractions (A—D) was diluted with a buffered aldehyde mixture of high osmolarity (KARNOVSKY, 1965) to 1.0 ml. These suspensions were placed separately on millipore filters of 0.22 p pore size and adhered to the surface in a holder with an inner diameter of 13 mm (Analysenfilterhalter, Millipore GmbH) by a water pump at 40—60 Hgmm vacuum. Filters were post-fixed in the same fixative for 2 hours at 4 °C. After overnight washing in the buffer used to make up the fixative, the filters were osmicated for 1 hour in buffered 1.0 per cent solution (MILLONIG, 1961) and dehydrated in graded ethanols. The filters were dissolved in propylene oxide, resulting in a thin layer of particles to be embedded flat. Sections were cut on a Porter-Blum ultramicrotome in a plane parallel to the direction of sedimentation and stained by lead citrate. Photographs were taken on a JEOL I00B electron microscope equipped with a side entry goniometer. 6 * 84 JO0, F. and RAKONCZAY. Z. Results and discussion Figures I and 2 show the electron microscopic appearance of the entire thickness of the fractions. In order to orientate the thin layer of fractions in the electron micros-cope. a BSR specimen holder was used, rendering possible rotation up to 180°. The surface facing the millipore filter, upon which particles were accumulated in a random distribution, could be easily recognized. The fine granular network, seen occasionally, may have resulted from soluble proteins. Fine structural features, characteristic of the myelin, synaptosomes. dendritic profiles and capillaries, could be readily iden-tified. Fig. I. A characteristic detail f rom the B (nerve ending) fract ion. N E = nerve ending. D = dendri te . My = myelin. x25 000. Millipore filters have already been introduced in electron microscopy by BAUD-HUIN et al (1967) for the preparation of very thin pellicles of packed particles. To accelerate the filtration of suspensions derived from rat liver, a special Filterfuge assembly was built and gas pressure of 3.6 kg c m - 2 was applied over the solution. It is well-known that the interpretation of biochemical and physiological experi-ments involving subcellular fractions depends considerably on the purity of the fractions (COTMAN and FRANSBERG, 1 9 7 0 : GROVE et al, 1973) . The main advantage of the simple sampling technique described here is that, apart from providing repre-sentative electron micrographs of fractions, it allows the carrying out of rapid quantitative evaluations on the relative concentration of an organelle in certain brain fractions. A S I M P L E S A M P L I N G T E C H N I Q U E 8 5 Fig. 2. Fine slruclural appearance of a capillary in Ihe D fraction. N = nucleus. L = l u m e n , B M = basement membrane. x l 8 000. References B A U D H U I N , P . . E V R A R D , P . and B E R T H E T , J . ( 1 9 6 7 ) : Electron microscopic examination of subcellular fractions. I. The preparation of representative samples from suspensions of particles. — J. Cell Biol. 32, 1 8 1 — 1 9 1 . C O T M A N , C . W. and F R A N S B F R G . D. A. (1970): An analytical micro-method for electron microscopic study of the composition and sedimentation properties of subcellular fractions. — Brain Res. 22, 152—156. G R O V E . W . E . , B O N D A R E F F , W . and VEIS , A . ( 1 9 7 3 ) : A sampling technique for quantitative electron microscopy of subcellular fractions. — J. Neurochem. 21, 7 0 3 — 7 0 4 . Joo, F . and K A R N U S H I N A , 1. ( 1 9 7 3 ) : A procedure for the isolation of capillaries f rom rat brain. — Cytobios, 8, 4 1 — 4 8 . KARNOVSKY, M. J. (1965): Formaldehyde-glutaraldehyde fixative of high osmolarity for use in electron microscopy. — J . Cell. Biol. 27, 137A. M I L L O N I G , G . (1961): Advantages of a phosphate buffer for OsO, solutions in fixation. — J. Appl. Phys. 32, 1637. Address of the authors : D R . F. J O 6 D R . Z . R A K O N C Z A Y Institutes of Biophyics and Biochemistry, Biological Research Center, H—6701 Szeged P. O. Box 521, Hungary 

A simple sampling technique for obtaining representative electron micrographs from brain fractions

http://acta.bibl.u-szeged.hu/21863/1/biologica_022_083-085.pdf

A simple sampling technique for obtaining representative electron micrographs from brain fractions

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