Histochemistry (1986) 85:251 253 Histochemistry 9 Springer-Verlag 1986 The application of lipid-soluble stains in plastic-embedded sections H. van Goor 1, P.O. Gerrits 2, and J. Grond 1 * Departments of 1 Pathology and 2 Anatomy, University of Groningen, Oostersingel 63, NL-9713 EZ Groningen, The Netherlands Received December 13, 1985 / Accepted February 1, 1986 Summary. The present study was designed to develop a routine method for direct demonstration and precize local- isation of lipid substances in tissue sections. A panel of lipid-rich tissues was fixed in 4% buffered formaldehyde, infiltrated, and embedded in the water-soluble plastics Technovit 7100, EFL-67, and JB-4. The use of alcohol con- taining fluids was avoided. Staining with the lipid-soluble dyes Sudan Black B and Oil Red O revealed excellent pres- ervation of tissue lipids in Technovit 7100 embedded sec- tions when compared with cryostat sections of the same tissue specimens. Lipid preservation in EFL-67 and JB-4 embedded sections was inconsistent, even when infiltration and polymerization procedures were performed at 4 ~ C. Combination of lipid-soluble dyes with the periodic acid Schiff, Jones' methenamine silver, or Gomori' reticulin method allowed for an exact localization of lipids in high- quality Technovit 7100 embedded sections. The procedure herein is easily applicable in routine histopathology prac- tice. Introduction The first stage in tissue processing of fixed material in rou- tine histopathology practice involves the removal of aque- ous tissue fluids by a variety of components, many of which are alcohols of varying types (Drury and Wallington 1980; Bancroft and Stevens 1982). In this dehydration process the alcohol soluble tissue lipids are extracted. Thus, in rou- tine paraffin and plastic sections (Ashley and Feder 1966; Rowden et al. 1982) lipids can only be recognized indirectly by the presence of optical lucent areas or vacuoles in which the presence of other tissue constituents such as mucus or glycogen is excluded by additional special stains. For direct demonstration of lipids, cryostat sections of fresh or forma- lin fixed material with a lower quality of morphology are required. The need for direct localization of lipids in tissue sections prompted us to apply water-soluble plastics as the solidifying medium with avoidance of alcohol containing fluids. Our method allows for the exact direct localization of tissue lipids in high quality plastic sections and is easily applicable in routine histopathology practice. * To whom offprint requests should be sent Material and methods Tissue processing. Small tissue specimens (4 x 4 • 2 ram) were ob- tained from atherosclerotic vessels, fatty liver, adrenal cortex, end- stage kidney, renal adenocarcinoma, and liposarcoma. They were fixed by immersion for 12 h to 4 weeks in 4% phosphate buffered formalin at room temperature. Three types of plastics were tested : JB-4 (Polysciences, Inc, Warrington, PA, USA) (Brinn and Pickett 1979; Horton et al. 1980; Poppema 1983), EFL-67 (Serva Feinbio- chemica, Heidelberg, Germany) (Nemetschek-Gansler et al. 1972) and Technovit 7100 (Kutzer & Co, GmbH, Bereich Technik, D- 6393 Wehrheim, Germany) (Gerrits and Smid 1983). After rinsing in phosphate-buffered saline for 2 h the tissue was infiltrated during a period of 2 h in two changes of 100% catalysed plastic. Infiltra- tion in Technovit 7100 was performed at room temperature, in EFL-67 and JB-4 at 4~ C. During this procedure the material was gently agitated in a vertical rotary motion, using a commercial mechanical device (GFL, Gesellschaft fiir Labortechnik mbH, D- 3006 Burgwedel, FR Germany). Specimens processed in Technovit 7100 were embedded in a mixture of 10 parts catalyzed Technovit 7100 infiltration solution (consisting of 40 ml Technovit 7100 (gly- col methacrylate, containing Co catalyst XCL), 4 ml of polyethyl- ene glycol 400, and 0.5 g of benzoyl peroxide (moistened with 20% H20)) and 1 part Technovit 7100 hardener II (the accelerator solu- tion, containing a barbituric acid derivate) (Gerrits and Staid 1983). Tissues processed in EFL-67 were embedded in a mixture of 20 parts catalyzed EFL-67 solution A (consisting of 80 ml glycol meth- acrylate stabilized with hydroquinone, 16 ml 2-bntoxyethanol, and 0.5 g benzoyl peroxide) and 1 part of the accelerator EFL-67 solu- tion B (consisting of 15 ml polyethylene glycol - carbowax 200 or 400 - and 1. ml of N,N-dimethylaniline) (Nemetschek-Gansler et al. 1972). Tissues processed in JB-4 were embedded in 25 parts catalyzed JB-4 solution A (consisting of 80 ml glycol methacrylate stabilized with hydroquinone, 16 ml 2-butoxyethanol, and 0.9 g benzoyl peroxide) and 1 part of the accelerator solution B (con- sisting of 15 ml polyethylene glycol 400 and 1 ml of N,N-dimethyl- aniline) (JB-4 Embedding Kit TM 1976 Data sheet 123, Polysci- ences Inc). Paraffin was poured around the blockholders placed in EFL-67 and JB-4, because the polymerization of these plastics is more susceptible to inhibition by oxygen than in the case of Technovit 7100 (Gerrits and Zuideveld 1983). Polymerization of Technovit 7100 was accomplished at room temperature in 3 h, of JB-4 and EFL-67 at 4~ in 3 h. Sections were cut at 2 gm on a Sorvall microtome with glass knives; stretched on a waterbath (23 ~ C), transferred to slides, and dried at room temperature. To check tissue lipid preservation in the plastic sections, 2 gm cryostat sections from the same tissue samples were used. Staining methods. Tissue lipids were demonstrated with the Oil Red O or Sudan Black B method. For staining with Oil Red O the sections were briefly rinsed in 60% isopropanol and stained