Staining histological lung sections with Sudan Black B or Sudan III for automated identification of alveolar epithelial type II cells

Abstract

Alveolar epithelial type II (AE2) cells produce, store and secrete pulmonary surfactant and serve as progenitor cells for the alveolar epithelium. They are thus an interesting target in wide fields of pulmonary research. Stereological methods allow their quantification based on measurements on histological sections. A proper AE2 cell quantification, however, requires a method of tissue processing that results in little tissue shrinkage during processing. It was recently shown that a primary fixation with a mixture of glutaraldehyde and formaldehyde, postfixation with osmium tetroxide and uranyl acetate and embedding in glycol methacrylate fulfills this requirement. However, a proper quantification, furthermore, requires a secure identification of the cells under the microscope. Classical approaches using routine stainings, high magnifications and systematic uniform random sampling can result in a tedious counting procedure. In this article we show that Sudan Black B and Sudan III staining in combination with the previously described “low shrinkage method” of tissue processing result in good staining of lamellar bodies of AE2 cells (their storing organelles of surfactant) and thus provide a good signal of AE2 cells, which allows their easy and secure identification even at rather low magnifications. We further show that this signal enables automated detection of AE2 cells by image analysis, which should make this method a suitable staining method for the recently developed and more efficient proportionator sampling.