The chensical nsechanisno of tise bins!ing of Congo red i)y aniyloisl is poorly unslerstoosi (10). In tissue sectiomss stained with Congo red in aqueous on alcoholic solutions w-ithount siifft-rentiation, only ansyloisi is dichroic. This ohservation insiicates a ussode of attao-hnst-nst of Congo red to ansyloid slifferent from the reas-tion of this dye svith other tissue structures. Furthermore, the polarization nsicroscopic ProPertit-s of antyloid stained with Congo red (6, 7, 10, 21, 29, 34) are strikingly sinsilar to those described for cellulose fibers slvesl with Congo ret! (1 1 , 32, 36, 37). The simusilanity of amyioisl ansi s-elltnlose was ensphasizeoi PUCHTLER, SWEAT AND LEVINE untes to 15 imotnrs. 5. Stairs ins Congo red solution for 20 minutes. Fonnumula: Stock solution: 80% ethyl alcohol sat-urnoteci with Congo red ansi NmuCl. Working solutions: Add 0.5 rum! 1% aslucous NaOH to 50 nmsl of the stock solution, filter. Use within 15 nmintntes.

Alizarin (madder) has been used in textile dyeing since early antiquity. In histology calcium-alizarin or calcium-alizarin red S compounds are often referred to as "lake" or "complex." Chemical and infrared spectroscopic data showed that these compounds are salts, not chelates. In dye chemistry the term lake denotes a poorly soluble or insoluble salt of a water-soluble dye. Salt formation between calcium deposits in tissues and alizarin or alizarin red S is indicated by the sensitivity of these compounds toward dilute acetic acid. Acid dyes for lakes, which do not contain chelating groups, also stained calcium deposits selectively. Alizarin stained calcium deposits intensely only around pH 12. Alizarin red S colored calcium deposits selectively around pH 9; neutral and acid dye solutions produced severe diffusion artifacts. Chemical data indicate that alizarin red S can react with calcium via its sulfonic acid and/or its OH groups.

Formalin has been recommended as an innocuous fixative for immunohistochemistry. However, several studies demonstrated impairment or blocking of antigenic activity of certain proteins. Formalin fixation was discovered accidentally by F. Blum in 1893 and its deleterious effects on various tissue structures were discussed extensively during the following decades. More recently, some authors assumed that formaldehyde bound to tissues can be largely or completely removed by washing and dehydration. According to chemical data, formaldehyde forms highly reactive methylols with uncharged amino groups. Such methylol groups yield methylene bridges with suitably spaced amides, arginine and aromatic amino acid sidechains. Only loosely bound formaldehyde is removed by washing for several hours. Residual bound formaldehyde cannot be dislodged by washing for weeks, but some formaldehyde is gradually removed when tissues are stored in water for an extended number of years. Methylene crosslinks resist treatment with high concentrations of urea, and can be broken only by drastic hydrolysis. It appears unlikely that such firmly bound formaldehyde is removed by conventional washing and dehydration procedures used in histochemistry. The superiority of methacarn, alcohol or acetone over formaldehyde fixation for immunohistochemical demonstration of prekeratin, myosin, type I and type IV collagen, laminin and fibronectin can be ascribed to the irreversible alterations of tissue proteins by formaldehyde.

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It has been suggested that in von Kóss'as technic silver cations replace calcium bound to phosphate or carbonate groups and are then reduced to black metallic silver during exposure to light. However, in test tube experiments silver phosphate retains its yellow color for days. These differences between reactions of pure calcium phosphates and calcium deposits in tissues were emphasized already by von Kóssa; he regarded only the initial yellow coloration of calcium diagnostic for calcium phosphates and deplored the subsequent blackening caused by organic compounds. Von Kóssa's experiments were easily reproducible. A review of the literature showed that reduction of silver nitrate by organic compounds was well known in the 19th century. For histochemical studies of phosphates it was deemed desirable to avoid the formation of black by-products. Sections of paraffin-embedded human tissues were exposed to solutions of silver nitrate in subdued light or darkness then treated with sodium thiosulfate. Silver phosphate was yellow to yellowish brown; other tissue structures remained colorless. No darkening was observed in sections stored for eight years. Other compounds which form yellow silver salts, e.g. iodides and periodates, are unlikely to occur in paraffin sections of human tissues.

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