Human intestinal mucosal mast cells: evaluation of fixation and staining techniques.

Abstract

SUMMARY The staining properties of tissue mast cells are influenced by the method of fixation. Differences in fixation and staining techniques may explain the contradictory results in the published reports on the number of human mucosal mast cells (MMC) in the gastrointestinal mucosa in health and disease.We have examined the influence of fixatives on the staining properties of human MMC in operative biopsy specimens of human jejunum. Specimens were divided into pieces, each of which was fixed in one of the following fixatives: Carnoy's, basic lead acetate (BLA), Baker's, Bouin's, isotonic formol-acetic-acid (IFAA), 10% neutral buffered formalin, formol sublimate, and formol saline.Thereafter, tissues were paraffin-embedded and 5 [km sections were cut and stained with either astra-blue/safranin pH 0-3, or Material and methodsOperative biopsies of normal jejunum were obtained from 13 patients who were undergoing abdominal surgery. In all cases the intestine was incised for resection or for creation of an anastomosis. Diagnoses were pyloric stenosis 4; adenocarcinoma of stomach or colon 3; duodenal ulcer 3; gastric ulcer 1; adhesions 1; unexplained abdominal pain 1. Each specimen was rinsed in cold saline and divided into eight pieces which were placed in fixative within 10 min. The period of fixation varied from one to seven days (except for Carnoy's) as indicated below. After fixation the tissues were embedded in paraffin; non-serial sections, 5 ,um thick, were cut and stained with astra-blue/safranin and with toluidine blue. Toluidine blue pH 0-54 Toluidine blue powder (0 5 g) (Gurr Chemical, Code Number 29880) was dissolved in 100 ml 0-5 N HCI (pH 0-5). The sections were taken to water as above and were stained with this solution for 30 min. They were rinsed in water for 5-10 min, differentiated in 95 % alcohol, and were cleared and mounted as above. METHOD FOR MAST CELL COUNTSMast cells were counted in well orientated sections cut perpendicular to the mucosa and in which the muscularis mucosae was intact. Counts were per- formed on coded slides on a Leitz Dialux 20 EB microscope (eyepiece x 10, objective x 40). A 10 mm2 eyepiece graticule, calibrated against a calibration slide was used. The edge of the graticule was orientated along the muscularis mucosae, at the base of the crypts. The area covered by the square of the graticule comprised 80-100 % of the total depth of the mucosa (from the bottom of the crypt to the tip of the villus) (Fig. 1). On each slide, eight to ten fields (290 ,um x 290 /tm; area 0 084 mm2) were counted and the MMC count per specimen was expressed as MMC/mm2. No attempt was made to correct for the area covered by epithelium. STATISTICAL ANALYSISResults are expressed as mean (x) and standard deviation (SD) or standard error of the mean (SE) and were compared by Student's t test. The reproducibility of duplicate counts on the same slide was assessed by linear regression analysis. ResultsOf the specimens obtained from the patients, 208 pieces of tissue were available for study, an...

Natural Schistosoma mansoni infection in Nectomys squamipes: histopathological and morphometric analysis in comparison to experimentally infected N. squamipes and C3H/He mice

“…Sections (5 µm) were stained with hematoxilin and eosin (H&E); PAS-Alcian Blue, pH = 1.0 and 2.5; Lennert's Giemsa (Lennert 1978); Gomori's reticulin stains, Sirius red pH = 10.2 (eosinophils) (Bogomoletz 1980, Luque & Montes 1989, Vale et al 1997); Masson's trichrome, Alcian blue/safranin (mast cell subpopulation) (Strobel et al 1981) and Picrosirius ± polarization microscopy (Junqueira et al 1979) and Weigert resorcin-fuchsin ± oxidation with potassium peroxymonosulfate (Oxone® monopersulfate compound, Sigma 22,803-6). Digital images were captured with Hamamatsu color chilled 3 CCD camera (model C-5810), stored with tagged-image file format (TIF) and printed in Codonics NP-1600 Photographic dye-sublimation Printer.…”

91166185 The ultrastructure of mast cells in the uterus throughout the normal menstrual cycle and the postmenopause

“…With increasing evidence of inter-and intra-species mast cell heterogeneity (Enerback 1966 a, b, c;Steer, 1976;Strobel, Miller & Ferguson, 1981) human mast cell function can no longer be inferred from studies using purified rat peritoneal mast cells. Cyclical changes and patient variability have been observed by light microscopy for mast cell numbers in the functional endometrium, basal endometrium and the endometrial/myometrial border throughout the menstrual cycle (Drudy, Sheppard & Bonnar, 1990 a).…”

Omental and pleural milky spots: different reactivity patterns in mice infected with Schistosoma mansoni reveals coelomic compartmentalisation

“…Whole omentum, lungs and the entire mediastinum of each animal were fixed in Carson's modified Millonig's phosphate-buffered formalin, pH 7.4 (Carson's formalin Millonig) (Carson et al 1973) for 48 h, with one change of the fixative at 24 h. Afterward, the samples were embedded in paraffin and histological sections (5 µm) were stained with Hematoxylin-Eosin, Lennert's Giemsa (Lennert 1978), Gomori's reticulin, Masson's trichrome, Weigert's resorcin-fucsin, PAS-Alcian Blue (pH 1.0 and 2.5) and Picrosirius red (with/without polarisation) (Junqueira et al 1979), Alcian-Blue safranin (Strobel et al 1981) and Sirius red (pH 10.2) (Bogomoletz 1980, Luque & Montes 1989. Sections stained with phosphomolybdic acid-picrosirius red (Dolber & Spach 1993) were analysed by confocal laser scanning microscopy (Zeiss LSM-410).…”