Bandeiraea simplicifolia lectin demonstrates significantly more capillaries in rat skeletal muscle than enzyme methods.

Determination of capillary density in skeletal muscle is often made after alkaline phosphatase staining [e.g.. Romanul (5)]. This procedure, however, reveals only the arterial portion of the capillary network, the venous portion remaining unstained (4). A more accurate measure of capillanity depends, therefore, on the ability to demonstrate both arterial and venous capillaries in the same preparation. This can be achieved by combining the methods for alkaline phosphatase and dipeptidyl peptidase IV (DPP IV); the latter is associated with venous capillaries (4). Capaldi et at. (1), cxamining the binding ofa range oflabeled lectins in skeletal muscle, noted that the tectin from Bandeiraea simplicifolia (BS1) selectively stained capillanes and the luminal surface of larger vessels. This finding was exploited by Hansen-Smith et at. (3), who used the TRI1t derivative of the tectin to identify capillaries and muscle fiber types simultaneously. In their abstract, these authors observed that the lectin revealed more capillaries than did alkaline phosphatase. This suggests that the lectin demonstrates both arterial and venous capillaries. To test this and the effectiveness of the 1cctin method, sections ofmuscle were stained with TRI1t-labeled lectin, photographed, and then reacted for DPP IV followed by alkaline phosphatase. The staining regimen was as follows: fresh 10sm transverse sections of rat soleus were cut in a cryostat, fixed for 15 mm in ice-cold acetone, hydrated to PBS, then stained for 1 hr at room temperature in 40-100 sg/ml TRI1tlabeled lectin (Sigma; London, UK) in PBS containing 1 mM CaCl2. The sections were then rinsed briefly in PBS, mounted in PBS, and photographed on a Leitz photomicroscope with a Ploemopak using the N2 filter block (530-560 nm). After a briefrinse in 0.1 M cacodytate buffer, pH 7.4, the sections were then incubated for DPP IV in the following freshly prepared medium for 30 mm at 37’C: 0.4 mg/mI Gly-Pro-4-methoxy-2-naphthylamide and 0.4 mg/mI 3,3’-dimethoxybenzidine fluoroborate (6) in 0.1 M cacodylate buffer, pH 7.4. After rinsing in Tris buffer, pH 9.4, the sections were transferred to the alkaline phosphatase substrate for 15 mm at 37’C, consisting of the following: 0.2 mg/mI 5-bromo-4-chloro-3-indolyl phosphate and 0.7 mg/mI Tetranitro Blue Tetrazolium (TNBT). The sections