No abstract

Application of morphometric analysis to the study of neuromuscular junction is helpful to the quantitative approach of ultrastructural changes. In this study, twelve autopsied subjects of different ages were examined on the ultra‐structure of neuromuscular junctions using morphometric analysis. The postsynaptic area and postsynaptic membrane length were significantly greater in adults than in infants. The results indicate that the number and/or depth of the secondary synaptic clefts of adults are greater than those of infants. In the aged subjects, presynaptic membrane length and postsynaptic membrane density showed a significant decrease. The latter observations were thought to be the results of regressive changes of pre‐ and postsynaptic structure with aging. The variations of each structural element were also studied. The large variations were found in the postsynaptic area, postsynaptic membrane length, and membrane length ratio of adults and aged group. On the other hand, variations in postsynaptic membrane density were small in all age groups. The importance of the knowledge of these age related changes and variations in the studies of neuromuscular junction was discussed. ACTA PATHOL. JPN. 34: 1243–1249. 1984.

Mast cells in the mucosa of the gastro-intestinal tract are called mucosal mast cells. These cells are distinguished from connective tissue mast cells in the skin and serosal cavities in terms of their ultrastructural morphology, histochemistry, and responses to histamine liberators and growth factors (2,3,6,7,11) . The stainability of mucosal mast cells are well preserved after fixation in Carnoy's fixative or basic lead acetate, but not after formalin fixation (6,16) . The mucosal mast cells are stained by alcian blue, but not by safranin or berberine (10, 16), which stains connective tissue mast cells.Recently, avidin-biotin-peroxidase complex (ABC) has been reported to bind nonspecifically to connective tissue mast cells (1,4,15). We have observed that ABC consistently bound to skin mast cells with sharp contrast, but not to mucosal mast cells in rat small intestine (1). On the other hand, alcian blue pH 1.0 or 0.5 stains both skin and mucosal mast cells. To differentiate both types of mast cells, we have attempted to stain these cells using both the alcian blue and ABC method, and have found that an alcian blue-PAS-ABC consecutive staining gave satisfactory results. The PAS method was used as a counter stain.Six male SD rats, 7-10 weeks old, were anesthetized and then sacrificed by exsanguination. Skin from groin, ear, ileum, and bone marrow from femur were dissected out from the animals and fixed in Carnoy's fluid. These tissues were embedded in paraffin and cut at 5µm. The three staining methods were carried out as follows : Alcian blue method : 1. Place in 1 % alcian blue 8GX (J. T. Baker Chemical Co.) in 0.1 N HCl (pH 1.0) or 0.7 N HCl (pH 0.5) for 30 min. 2. Rinse in several changes of 0.1 N HCI. PAS method: 1. Place in 1 % periodic acid for 10 min. 2. Rinse in running tap water for 5 min. 3. Place in Schiff's solution for 10 min. 4. Place in running tap water for 10 min. ABC method: 1. Place in 3 % H2O2 for 5 min. 2. Rinse in three changes of 0.05 M Tris buffer solution pH 7.6. 3. Incubate with avidin-biotin-peroxidase complex (Vectastain ABC kit, Vector Lab.) for 30 min at room temperature. 4. Rinse in three changes of the buffered solution. 5. Incubate in 0.024% DAB, 0.005% H2O2 in the buffered solution for 5 min. 6. Rinse in the buffered solution. After staining, sections were dehydrated through graded ethanol series, cleared in xylene and mounted in a synthetic resin.For the combined staining, we examined all possible sequences of alcian blue, ABC and PAS. With every sequence, intestinal mucosal mast cells stained only blue, whereas skin mast cells exhibited a mixed color of blue and brown. However, the staining of intestinal mucosal mast cells with alcian blue was always weak in the sequences where ABC and/or PAS were performed prior to alcian blue. Further, diffusion of ABC staining products was sometimes observed with these sequences. On the other hand, alcian blue-PAS-ABC or alcian blue-ABC-PAS sequence gave satisfactory mast-cell staining with alcian blue. Lev and Spicer (8) reported that st...