0047-7206/82/030369-02503.00/0 Micron, Vol.]3, No.3, pp.369-370, 1982. Printed in Great Britain Pergamon Press Ltd.

CRYOSTAT SECTIONING TO EXPOSE THE LUMINAL SURFACE OF BLOOD VESSELS FOR SCANNING ELECTRON MICROSCOPY

K. Yamaguchi and G.I. Schoefl

Department of Experimental Pathology, John Curtin School of Medical Research The Australian National University, Canberra, ACT 2601

We have recently examined high endothelium venules in Peyer's patches of the mouse intestine. In these modified blood vessels, large numbers of recirculat- ing lymphocytes migrate across the vascular wall. We wished to examine the luminal surface of these vessels with the scanning electron microscope and to analyse the sites where lymphocytes penetrate the endothelium. This required that long stretches of the inner vascular surface be available for observa- tion. Since blood vessels associated with Peyer's patches form networks which are oriented roughly parallel to the gut wall, cleavage of the tissue in that plane was desirable.

The intestine was perfused with a 2% glutaraldehyde/2% formaldehyde mixture in 0.I M sodium cacodylate via the thoracic aorta and excised segments containing a Peyer's patch were immersed in the same fixative overnight. They were then impregnated with osmium I before attempts were made to expose blood vessels deep in the tissue. (a) Fracturing under liquid nitrogen: Fractures across the gut wall were obtained easily with the method of Humphrey et al. 2 However, only short segments or cross-sections of blood vessels were exposed in that plane (Fig. i). Attempts to fracture the tissue parallel to the gut wall were unsuccessful. Trials included clamping the Peyer's patch area between two SEM specimen stubs and either pulling the stubs apart under liquid nitrogen, or fracturing the wedged tissue by tapping it with a razor blade. Better results were obtained when slivers of tissue were carefully chipped away from the outer surface of the gut with a razor blade held in artery forceps (Fig. 2) though the plane of section was difficult to control. (b) Cryostat: Areas of Peyer's patch were frozen onto a cryostat specimen holder in a drop of buffer and then sectioned parallel to the gut wall. The level to which tissue had been removed from either the serosal or the mucosal surface was monitored in the sections. When a suitable level had been reached, the tissue was dehydrated in acetone, critical point dried and coated with gold. Figures 3 and 4 illustrate the long segments of longitudinally sectioned blood vessels which could be obtained with this method. As previously reported by other workers 3'~, freezing had no deleterious effect on the tissue. Similar results were obtained with tissues impregnated with camphene which were mounted on the cryostat holder in camphene and subsequently dried in vacuo 5.

REFERENCES

i. T. Murakami, Arch. histol. Jap. 36:189-193, 1974 2. W.J. Humphrey, B.O. Spurlock and--~.S. Johnson, Scanning Electron

Microscopy I:275-282, IIT Res. Inst., Chicago, 1974 3. M. Carri and A. Suburo, Anat. Rec. 193:857-862, 1979 4. D.K. Dueber, H.A. Chaudhry and D.J. Pihlaja, Scanning Electron Microscopy

III:323-328, SEM Inc., AMF O'Hare, 1979 5. W.B. Watters and R.C. Buck, J. Microscopy 94:185-187, 1971

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Fig. Transverse cryofracture of the gut wall. HEV = high endothelium venule.

Fig. 2 An arborizing venous segment. Overlying tissue has been chipped away with a razor blade.

Fig. 3 Low power of a Peyer's patch planed with a cryostat to expose the venous network.

Fig. 4 Higher magnification of a high endothelium venule shown in Figure 3.