Abstract
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.
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References
Larson PS, De Las Morenas A, Cerda SR, Bennett SR, Cupples LA, Rosenberg CL (2006) Quantitative analysis of allele imbalance supports atypical ductal hyperplasia lesions as direct breast cancer precursors. J Pathol 209:307–316
Heaphy CM, Griffith JK, Bisoffi M (2009) Mammary field cancerization: molecular evidence and clinical importance. Breast Cancer Res Treat 118:229–239
Lakhani SR, Chaggar R, Davies S et al (1999) Genetic alterations in “normal” luminal and myoepithelial cells of the breast. J Pathol 189:496–503
Tot T (2007) The theory of the sick lobe and the possible consequence. Int J Surg Pathol 15:369–375
Kauff ND, Brogi E, Lauren S, Pathak DR, Borgen PI, Hudis CA, Offit K, Robson ME (2003) Epithelial lesions in prophylactic mastectomy specimens from women with BRCA mutations. Cancer 97:1601–1608
Going JJ, Moffat DF (2004) Escaping from Flatland: clinical and biological aspects of human mammary duct anatomy in three dimensions. J Pathol 203:538–544
Tot T (2005) DCIS, cytokeratins, and the theory of the sick lobe. Virchows Archiv 447:1–8
Wellings SR, Jensen HM, Marcum RG (1975) An atlas of subgross pathology of the human breast with special reference to possible precancerous lesions. J Natl Cancer Inst 55:231–273
Kordon EC, Smith GH (1998) An entire functional mammary gland may comprise the progeny from a single cell. Development 125:1921–1930
Love SM, Barsky SH (1996) Breast-duct endoscopy to study stages of cancerous breast disease. Lancet 348:997–999
Murata S, Kominsky SL, Vali M et al (2006) Ductal access for prevention and therapy of mammary tumours. Cancer Res 66:638–645
Going JJ, Mohun TJ (2006) Human breast duct anatomy, the “sick lobe” hypothesis and intraductal approaches to breast cancer. Breast Cancer Res Treat 97:285–291
Going JJ, Moffat DF (1996) Three dimensional anatomy of complete duct systems in human breast: pathological and development implications. J Clin Pathol 49:48–52
Armstrong JS, Davies JD, Hronkova B (1992) Backprocessing paraffin wax blocks for subgross examination. J Clin Pathol 45:1116–1117
Landau JD, Visbal AP, Lewis MT (2009) Methods for preparing fluorescent and neutral red stained whole mounts of mouse mammary glands. J Mammary Gland Biol Neoplasia 14:411–415
Staudt T, Lang MC, Medda R, Engelhardt J, Hell SW (2007) 2,2′-Thiodiethanol: a new water-soluble mounting medium for high resolution optical microscopy. Microsc Res Tech 70:1–9
Appleton PL, Quyn AJ, Swift S, Nathke I (2009) Preparation of wholemount mouse intestine for high-resolution three dimensional imaging using two-photon microscopy. J Microsc 234:196–204
Animals (Scientific Procedures) Act 1986, Sch 1.
Rasmussen SB, Young LJT, Smith GH (2000) Preparing mammary gland whole mounts from mice. In: Margot MI, Bonnie BA (eds) Methods in mammary gland biology and cancer research. Kluwer Academic, New York, pp 75–85
Manton SL, Ferguson DJ, Anderson TJ (1981) An automated technique for the rapid processing breast tissue for subgross examination. J Clin Pathol 34:1189–1191
Samoilov VI, Chebyshev AP (1964) Whole mount preparations of rat and mouse mammary glands. Bulletin Exp Biol Med 58:1468–1469
Steinke H, Wolff W (2001) A modified Spalteholz technique with preservation of the histology. Ann Anat 183:91–95
Wells HG (1897) The invisible man. Pearson, London
Smith RM, Matiukas A, Zemlin CW, Pertsov AM (2008) Nondestructive optical determination of fiber organisation in intact myocardial wall. Microsc Res Technique 71:510–516
Cassot F, Lauwers F, Lorthois S, Puwanarajah P, Cances-Lauwers V, Duvernoy H (2010) Branching patterns for arterioles and venules of the human cerebral cortex. Brain Res 1313:62–78
Dabelow A (1957) Die Milchdrüse. In: Mollendorf WV, Bragmann W (eds) Handbuch der mikroskopischen Anatomie des Menshcen. Springer, Berlin, pp 1–277
Zucker RM (2006) Whole insect and mammalian embryo imaging with confocal microscopy: morphology and apotosis. Cytometry A 69:1143–1152
Howard MD, Cann M, Henry MS, Verhulst L (1958) The salicylate problem with special reference to methyl salicylate. J Paediatrics 53:271–276
Haberman J (1948) Clearing brain tissue with anethole. Biotech Histochem 23:143–144
Jain S, Khoury JM, Chamon W, Azar DT (1995) Corneal light scattering after laser in situ keratomileusis and photorefractive keratectomy. Am J Ophthalmol 120:532–534
Stockert JC, Llorente AR, Castillo PD, Gomez A (1990) Chromatin fluorescence after carmine staining. Biotech Histochem 65:299–302
Megalooikonomou V, Barnathan M, Kontos D, Bakic PR, Maidment ADA (2009) A representation and classification scheme for tree-like structures in medical images: analyzing the branching pattern of ductal trees in X-ray galactograms. IEEE Transactions Med Imaging 28:487–493
Clarke GM, Peressotti C, Constantinou P, Hosseinzadeh D, Martel A, Yaffe MJ (2011) Increasing specimen coverage using digital whole-mount breast pathology: implementation, clinical feasibility and application in research. Computerized MedicalImaging and Graphics 35:531–541
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This work was supported by the School of Medicine of the University of Glasgow.
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Smith, W.A., Going, J.J. Subgross breast pathology in the twenty-first century. Virchows Arch 460, 489–495 (2012). https://doi.org/10.1007/s00428-012-1226-y
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DOI: https://doi.org/10.1007/s00428-012-1226-y