Alcian Blue and Pyronine Y histochemical stains permit assessment of multiple parameters in pulmonary disease models

Abstract

Utilization of a combined Alcian Blue and Pyronine Y histochemical method for the assessment of multiple parameters in the respiratory tract of various species is described. Acidic mucins were deep blue (sialylated mucins), red (sulfated mucins), or variably purple (mixture of sialylated/sulfated mucins), and differential mucus production was readily detected in a murine respiratory syncytial virus vaccine model of pulmonary inflammation. Elastic fibers stained red in the walls of pulmonary arteries, connecting airways, alveolar septa, and subpleural interstitium. Mast cells had red to red-purple granular cytoplasmic staining. Nuclei were ubiquitously counterstained pale blue. Representative staining was detected in tissues from multiple species, including inbred mice, rats, ferrets, cats, dogs, sheep, and pigs. The fluorescent property of the stained tissues offers additional modalities with which to analyze tissue sections. This histochemical technique detects multiple critical parameters in routine paraffin sections of lung tissue, reduces the need for repeated serial sectioning and staining, and is cost-effective and simple to perform.

Figure 1

Fig. 1. Proximal trachea; BALB/cNCr mouse. The tracheal cartilage stains red with mucus stained variably blue to red-purple in proximal tracheal submucosal glands (arrows). Alcian Blue/Pyronine Y. Bar = 0.5 mm. Fig. 2. Lung; BALB/cNCr mouse. Peribronchial mast cells (arrows) have red granular cytoplasmic staining. Alcian Blue/Pyronine Y. Bar = 50 µm. Fig. 3. Lung; BALB/cNCr mouse. Airway from a beta-galactosidase-primed RSV infected mouse with little to no blue staining of airway epithelium. Alcian Blue/Pyronine Y. Bar = 100 µm. Fig. 4. Lung; BALB/cNCr mouse. Airway from a RSV G-protein-primed RSV-infected mouse. Note the intense deep blue mucus detection (arrows) in connecting airway epithelium. Alcian Blue/Pyronine Y. Bar = 100 µm. Fig. 5. Lung; Pig. Normal bronchus with submucosal gland and surface epithelial mucus staining magenta. Periodic Acid Schiff (PAS). Bar = 50 µm. Fig. 6. Lung; Pig, serial section of Figure 5. Overall staining pattern is similar to Figure 5 (PAS), but sialylated (blue), sulfated (red) or mixed (purple) mucus can be differentiated. Alcian Blue/Pyronine Y. Bar = 50 µm. Fig. 7. Lung; Cat. Arrows indicate elastic fibers subjacent to bronchial epithelium. Bar = 50 µm. Fig. 8. Lung; Cat. Chronic bronchitis with severe bronchiectasis demonstrating qualitative change in mucus expression (increased red staining) and a lack of elastic fibers staining. Alcian Blue/Pyronine Y. Bar = 50 µm. Fig. 9. Artery; BALB/cN mouse. Elastic fibers stain red, Alcian Blue/Pyronine Y. Inset, Intense fluorescence of elastic fibers in the same section examined via fluorescent microscopy. Bar = Bar = 33 µm.