JOURNAL OF ELECTRON MICROSCOPY TECHNIQUE 18:209 (1991)
Rapid Communication A Fast, Simple Method for Preparing Flat Semi-thin Sections from Epoxy Embedded Materials R. E. Kingsley and Jaime Dant South Bend Center for Medical Education Indiana University School of Medicine South Bend, IN 46556 INTRODUCTION The use of semi-thin specimens, cut from plastic embedded material, is an important adjunct to electron microscopy and many methods for their preparation have bcen developed over the years. Unfortunately, when cut from epoxy embedded blocks these sections are very difficult to stain and frequently they d o not dry completely flat on the slide. Martins-Green offers one solution, but that method takes 24 hours to accomplish? While the use of water-soluble methacrylates has overcome these difficulties to a large degree, specimens embedded in this way are frequently not suitable for electron microscopy. In this paper we describe a very rapid and simple method for obtaining flat semi-thin sections from epoxy embedded materials. METHOD: The tissue is prepared in the usual manner. For corneal tissue, we fix the whole cornea in 2% glutaraldehyde buffered in 0.1M sodium cacodylate; post-fix it in 1%osmium tetroxide prepared in the same buffer; dehydrate over a graded series of ethanols; embed in Spurr's epoxy formulation (Ted Pella, Inc.) and polymerize at 6W€. We cut the sections with a diamond histology knife (Delaware Diamond Inc.) at 0 . 5 ~ to 1 . 5 depending ~ upon the nature of the tissue. We have had success with sections ranging in area from 0.5mm2 to 2.0mm2. We pick up the sections by slipping a broken piece of cover glass (5mm x 25mm) under the sections and teasing them to the water interface with a Dalmatian Dog hair'. Once one edge of the section has adhered to the glass, it is withdrawn from the water at an angle, leaving the section resting on a slight film of water on the cover glass. Alternatively we pick u p the sections in a platinum loop (Ted Pella, Inc.). Touching the loop to the cover glass leaves the section floating in a small droplet of water. The cover glass is held in a pair of self-closing watchmaker forceps and heated gently for about 3 seconds, 2 to 3 inches above an alcohol flame, until the glass beqns to fog. At this point, slowly wave it through the blue flame with a side-to-side motion. As the water continues to evaporate, watch the section carefully. Just as the section is about to touch the glass, hold the cover glass away from the flame, in the air as the water completes its evaporation. If you heat the section too long and get it too hot, the sections will develop a bubble which will either burst and destroy the section or wrinkle it beyond usefulness. At this point the sections can be viewed with phase contrast, modulation or differential interference optics, either dry (unmounted, face up to the microscope objective) or mounted with synthetic media (ProTcxx Mounting Medium - Scientific Products M7635-1). We have noticed that mounting media which contains toluene will wrinkle the sections several hours after they have been enclosed. Since most synthetic media contain large amounts of toluene, we allow the solvents to evaporate and then add xylene until the mixture is of a workable consistency. Modified in this way, it poses no threat to the sections. Tandle? suggests the use of unpolymerized resin as an ericlosing media. N O T E This work was supported in part by NIH grant EY05717 to C. F. Marfurt and K. E. Kingsley.
REFERENCES:
1. Forbes, M.S.; (1986) Hints and Tips: Dog Hairs as Section Manipulators. E M S A Bulletin 26:67. 2. Martins-Green, M.; (1900) A simple procedure to obtain thick flat cornea sections for optical microscopy.
Stain Technol 296-298.
3. Tandler, B.; (1990) Improved slides of semithin sections. ].Elecfron Microsc.Tech. 14:285-286. Received February 1, 1991; accepted February 6, 1991.
01991 WILEY-LISS, INC.
Rapid Communication A Fast, Simple Method for Preparing Flat Semi-thin Sections from Epoxy Embedded Materials R. E. Kingsley and Jaime Dant South Bend Center for Medical Education Indiana University School of Medicine South Bend, IN 46556 INTRODUCTION The use of semi-thin specimens, cut from plastic embedded material, is an important adjunct to electron microscopy and many methods for their preparation have bcen developed over the years. Unfortunately, when cut from epoxy embedded blocks these sections are very difficult to stain and frequently they d o not dry completely flat on the slide. Martins-Green offers one solution, but that method takes 24 hours to accomplish? While the use of water-soluble methacrylates has overcome these difficulties to a large degree, specimens embedded in this way are frequently not suitable for electron microscopy. In this paper we describe a very rapid and simple method for obtaining flat semi-thin sections from epoxy embedded materials. METHOD: The tissue is prepared in the usual manner. For corneal tissue, we fix the whole cornea in 2% glutaraldehyde buffered in 0.1M sodium cacodylate; post-fix it in 1%osmium tetroxide prepared in the same buffer; dehydrate over a graded series of ethanols; embed in Spurr's epoxy formulation (Ted Pella, Inc.) and polymerize at 6W€. We cut the sections with a diamond histology knife (Delaware Diamond Inc.) at 0 . 5 ~ to 1 . 5 depending ~ upon the nature of the tissue. We have had success with sections ranging in area from 0.5mm2 to 2.0mm2. We pick up the sections by slipping a broken piece of cover glass (5mm x 25mm) under the sections and teasing them to the water interface with a Dalmatian Dog hair'. Once one edge of the section has adhered to the glass, it is withdrawn from the water at an angle, leaving the section resting on a slight film of water on the cover glass. Alternatively we pick u p the sections in a platinum loop (Ted Pella, Inc.). Touching the loop to the cover glass leaves the section floating in a small droplet of water. The cover glass is held in a pair of self-closing watchmaker forceps and heated gently for about 3 seconds, 2 to 3 inches above an alcohol flame, until the glass beqns to fog. At this point, slowly wave it through the blue flame with a side-to-side motion. As the water continues to evaporate, watch the section carefully. Just as the section is about to touch the glass, hold the cover glass away from the flame, in the air as the water completes its evaporation. If you heat the section too long and get it too hot, the sections will develop a bubble which will either burst and destroy the section or wrinkle it beyond usefulness. At this point the sections can be viewed with phase contrast, modulation or differential interference optics, either dry (unmounted, face up to the microscope objective) or mounted with synthetic media (ProTcxx Mounting Medium - Scientific Products M7635-1). We have noticed that mounting media which contains toluene will wrinkle the sections several hours after they have been enclosed. Since most synthetic media contain large amounts of toluene, we allow the solvents to evaporate and then add xylene until the mixture is of a workable consistency. Modified in this way, it poses no threat to the sections. Tandle? suggests the use of unpolymerized resin as an ericlosing media. N O T E This work was supported in part by NIH grant EY05717 to C. F. Marfurt and K. E. Kingsley.
REFERENCES:
1. Forbes, M.S.; (1986) Hints and Tips: Dog Hairs as Section Manipulators. E M S A Bulletin 26:67. 2. Martins-Green, M.; (1900) A simple procedure to obtain thick flat cornea sections for optical microscopy.
Stain Technol 296-298.
3. Tandler, B.; (1990) Improved slides of semithin sections. ].Elecfron Microsc.Tech. 14:285-286. Received February 1, 1991; accepted February 6, 1991.
01991 WILEY-LISS, INC.
