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Preparation of rodent kidney for light microscopy by freeze‐drying and embedding in water‐soluble methacrylate
Author(s) -
MIDDLETON E.
Publication year - 1967
Publication title -
journal of the royal microscopical society
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.569
H-Index - 111
eISSN - 1365-2818
pISSN - 0368-3974
DOI - 10.1111/j.1365-2818.1967.tb04488.x
Subject(s) - methacrylate , methyl methacrylate , materials science , penetration (warfare) , chemical engineering , chromatography , chemistry , composite material , copolymer , polymer , operations research , engineering
SYNOPSIS Methods for the elimination of five main artifacts common to frozen‐dried specimens embedded in methacrylate are described in detail. Two of these are associated with the drying technique and three to the methacrylate embedding process. One of the “drying” artifacts, contamination of the tissues by pump oil, can be overcome by inserting an oil trap between the vacuum pump and the freeze‐dryer. The other, “zoning” in the specimens, is shown to be due to incomplete drying. Using an Edwards TD2 or TD4 freeze dryer a point in the drying process is reached when the pressure exerted by the water extracted from the specimens equals that exerted by the residual water within the specimens. Drying then ceases and cannot be completed merely by prolonging the procedure. To complete drying, the tissues are transferred to a second clean drying head and the process is repeated. Good impregnation of tissues is achieved by evacuating them in a chamber before adding the methacrylate, which is then allowed to penetrate the tissues at atmospheric pressure. This calls for a viscous methacrylate such as the glycol compound. The rate of penetration varies greatly for different tissues and methacrylates and a glycol/butyl methacrylate mixture penetrates frozen‐dried kidney very slowly at a rate of about 1 mm in twelve hours. The remaining artifacts are ballooning of the tissues, leaching out of material from them, and bubbling of the methacrylate. The properties of glycol, butyl, and methyl methacrylates, alone and in mixtures, have been studied in association with different catalyst concentrations. A mixture of 85 p.c. distilled glycol and 15 p.c. distilled butyl methacrylate with 0.5 p.c. benzoyl peroxide catalyst added, satisfies the vacuum embedding requirements, obviates ballooning of tissues and leaching out of material from them, does not bubble and produces a block with an acceptable cutting texture. It is necessary to polymerize some of the mixture to within ½ hour of completion for casting the final block.