Harris hematoxylin hhs 32

Whole lungs were inflated with 10% formalin (Fisher Scientific) and then fixed for ≥24 h in 10% formalin. Fixed lungs were embedded in paraffin (Leica), cut into 4 µm sections, and applied to glass slides. Slides were washed with xylene (Chaptec) and then immersed in ethanol for 10 min. Slides were rinsed with distilled water and then briefly submerged in 50% Harris hematoxylin (HHS-32; Sigma) diluted in distilled water. Slides were rinsed under running tap water, washed 10x with ethanol, and then stained with eosin-phloxin B [100 ml 1% eosin Y (Sigma), 10 ml phloxin B (Sigma), 780 ml ethanol, 4 ml glacial acetic acid (Fisher Scientific)]. Slides were immersed in ethanol for 10 min, dried, and then submerged in xylene for 10 min. Slides were fixed with two drops of acrytol (Leica) with a coverslip. Sections were stained with hematoxylin and eosin as previously described³² . Images were obtained using a Zeiss Primo Star light microscope equipped with an AxioCam ERc5s (Zeiss) camera.

Slides were thawed to room temperature (1 h) and fixed in 4% paraformaldehyde in 0.01 M PBS for 15 min, then rinsed in several washes of distilled water, and stained with Harris Hematoxylin (HHS32, Sigma-Aldrich) for 15 min. Sections underwent differentiation in 1% acid–alcohol, a quick bluing step in 0.2% ammonia water, and several water rinses. Slides were counter-stained with eosin Y (EOS109, BioShop). Sections were dehydrated by a series of graded ethanol, cleared by xylene, and cover-slipped using a solvent-based mounting medium (Cytoseal 60™, Thermo Scientific).