A combined Alcian blue-PAS-ABC method for differential staining of mast cells.

Abstract

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...

Lectin histochemistry of the mast cell: Heterogeneity of rodent and human mast cell populations

“…This is reflected in their different pattern of staining with Atcian Blue and Safranin (Enerback, 1966b), berberine sulphate (Enerback, 1974;Wingren & Enerbfick, 1983) and avidin peroxidase (Arizono et al, 1987;Roberts & Bell, unpublished data), and in the differential sensitivity of *To whom correspondence should be addressed. 0018-2214/90 $03.00 +.12 9 1990 Chapman and Hall Ltd. their staining to nitrous acid and variation of ionic strength (Enerback, 1987).…”