Subgross breast pathology in the twenty-first century

Subgross studies on breast morphology in cleared thick sections are informative, but lengthy protocols could clash with diagnostic timescales and interfere with immunohistochemistry (IHC) or molecular analysis. We sought to speed up staining and tissue clearing to improve compatibility with diagnostic needs without detriment to histology or other assays. Thick sections (2-3 mm) of normal human breast tissue and whole mouse mammary glands were stained with alum-carmine, aceto-carmine, Harris's haematoxylin, cresyl violet, neutral red, thionin, 4',6-diamidino-2-phenylindole (DAPI), ethidium bromide (EB), or propidium iodide (PI) and cleared in xylene (refractive index ν = 1.50), benzyl alcohol/benzyl benzoate (BABB; ν = 1.59), xylene-BABB (X-BABB), thiodiglycol (2,2'-thiodiethanol, ν = 1.52) or anethole (ν = 1.56). Tissue was then paraffin embedded for IHC for ER, PR, E-cadherin, CD31 or cytokeratin 7. Haematoxylin and alum-carmine are excellent non-fluorescent subgross stains giving strong nuclear staining and minimal background. DAPI and EB permeate thick sections poorly but PI penetrates well, with a high signal-to-noise ratio after clearing in BABB or X-BABB. Other clearing agents were less effective, including thiodiglycol. Anethole's unpleasant odour precluded further evaluation. All evaluated clearing agents preserved comparable immunoreactivity for all markers. PI is a promising stain for subgross breast studies, compatible with BABB clearing. The hope that the water-miscible thioalcohol, thiodiglycol, might accelerate tissue clearing was not realised. There is scope for further streamlining to make subgross techniques more acceptable in combined research/diagnostic settings.