Observations on Substances that react weakly to the Periodic Acid/Schiff Test By ARTHUR J. HALE (From the Histology Division, Institute of Physiology, The University, Glasgow) With one plate (fig. i) SUMMARY It has been shown that certain mutinous substances which are weakly positive with the periodic acid/Schiff technique stain more strongly after treating them with a solution of sodium hydroxide. The deepening of the staining reaction is produced by making available, for aldehyde production by periodic acid, more .CHOH.CHOH., .CHOH.CHNH2., or . CHOH .CHNHR. groups. This is done without affecting those sulphate or metaphosphate groups responsible for metachromasia. INTRODUCTION N histological sections there are present a number of substances loosely I described as being of a mucinous nature. Examples of these are the ground substance of cartilage and of other less dense connective tissues, sarcolemma, basement membranes, colloid of endocrine glands, and the mucus of epithelia. Histochemically they are related and the basis of the relationship is the presence of a carbohydrate found either as a separate entity or in combination with protein or lipide. The biochemistry of these substances is as yet incom- pletely understood, but there are available histochemical methods which give some indication of the composition of any one member of this family. One of these is the periodic acid/Schiff technique (McManus, 1946). Some of the substances mentioned above will give a strong colour reaction with this method, others will only give a weak reaction. Glycogen, the ground substance of cartilage, and certain types of mucus are examples of the former; other types of mucus and sarcolemma are examples of the latter. The periodic acid/Schiff (P.A.S.) technique (Lillie, 1947; Hotchkiss, 1948; McManus, 1948) is really a Malaprade reaction (Lillie, 1950). It identifies .CHOH.CHOH., .CH0H.CHNHa., and .CHOH.CHNHR. groups in polysaccharides, mucopolysaccharides, mucoproteins, glycoproteins, and glycolipides by production of aldehydes, through periodic acid oxidation, which colourize the Schiff aldehyde-reagent. Hale (1946), Gersh (1949), and Roberts and Jarrett (1950) have presented methods which may be of use in differentiating between substances which give a positive reaction with P.A.S. Hempelmann (1940), Friedenwald (1947), [Quarterly Journal of Microscopical Science, Vol. 94, part 3, pp. 303-313, Sept. 1953.] 304 Hale—Observations on the Periodic Acid\Schiff Test and Grishman (1948) have introduced methods for differentiating tissue constituents of this type which stain metachromatically. Dempsey and his associates (1946, 1947, and 1950) and Pearse (1949 and 1950) have usedmethods for identifying different mucopolysaccharides by their degree of basiphilia. This report is the result of the finding that pre-treatment of histological sections with a weak solution of sodium hydroxide accentuates, in some cases, the depth of staining-reaction obtained with the P.A.S. method. MATERIALS AND METHODS As interpretation of the results depended on intensity of the colour re- action which developed, care was taken to subject all sections to identical procedures. The periodic acid solution was prepared as described by Lillie (1947). The Schiff solution was prepared as suggested by Lillie (1951a) and modified to conform with the findings of Longley (1952) and Atkinson (1952) in order to make the results more easily reproducible. The sulphite rinse between periodic acid oxidation and immersion in Schiff's solution has been shown by Lillie (195 ib) to reduce the intensity of the staining reaction with periodic acid, and this step has been omitted through- out in order to maintain the strength of any possible positive reaction. Acetylation blocks .CHOH.CHOH., CHOH.CHNH2., and .CHOH. CHNHR. groups (Gersh, 1949; McManus and Cason, 1950) and prevents the production of aldehydes by subsequent periodic acid oxidation. De- acetylation returns the groups so blocked to their original potential reactivity. Acetylation of sections was carried out as described by Lillie (195 ib). It was found that deacetylation in 75 per cent, ethanol and ammonia was more efficient than that in absolute ethanol and ammonia as used by Lillie. Immersion of sections in hydroxylamine hydrochloride (Danielli, 1949), to block all aldehydes, was used to demonstrate the specificity of the Schiff's solution and to identify the nature of the reaction product. The method for the Feulgen reaction, in so far as it could be carried out in paraffin sections, followed the instructions of Danielli (1949). In all cases, batches of sections containing one slide from each tissue were carried through each procedure. Batches were placed in glass racks and times were accurately controlled. All sections, irrespective of the fixative used, were taken through iodine and thiosulphate when being brought down to water before staining. All solutions were renewed from standard stock bottles each day. Washing was carried out in running tap water. Glasgow tap water is very pure and constant in quality (Garven and Gairns, 1952) and no harm appears to be caused by washing sections in it. Before and after the periodic acid solu- tion the sections were washed in 70 per cent, ethanol. All sections were dehydrated in 90 per cent, ethanol and two changes of absolute ethanol, cleared in xylene, and mounted in D.P.X. Counterstains were not used. The tissues used were known to give a weakly positive P.A.S. reaction in at least part of their substance. They were: Hale—Observations on the Periodic AcidjSchiff Test 305 Human tissues fixed in 10 per cent, neutral formalin: heart (lipofuscin), cervix, prostate, rectum, pancreas, trachea, sub- maxillary gland; fixed in mercuric formalin: rectum, amyloid spleen, aorta, umbilical cord, amyloid liver, myxoma, heart valve; fixed in Bouin's fluid: lung (pneumonic exudate), umbilical cord, small intestine, stomach. Rabbit tissues fixed in 10 per cent, neutral formalin: duodenum, kidney; fixed in Bouin's fluid: bone marrow. Rat tissues fixed in 10 per cent, neutral formalin : eye, mesentery. Representative batches of sections were carried through by each of the following procedures after having been brought to water. Room temperature (approximately 200 C.) was used except where otherwise stated. (1) Periodic acid 15 min.; wash; Schiff soln. 30 min.; wash. (2) Schiff soln. 30 min.; wash. (3) o-2N NaOH 15 min. at 22° C.; wash; periodic acid 15 min.; wash; Schiff soln. 30 min.; wash. (4) o-2N NaOH 15 min. at 220 C; wash; Schiff soln. 30 min.; wash. (5) Acetylation; wash; periodic acid 15 min.; wash; Schiff soln. 30 min.; wash. (6) Acetylation; wash; deacetylation; wash; periodic acid 15 min.; wash; Schiff soln. 30 min.; wash. (7) o-2N NaOH 15 min. at 22° C; wash; acetylation; wash; periodic acid 15 min.; wash; Schiff soln. 30 min.; wash. (8) Acetylation; wash; o-2NNaOH 15 min. at22°C; wash; periodic acid 15 min.; wash; Schiff soln. 30 min.; wash. (9) Deacetylation 15 min.; wash; periodic acid 15 min.; wash; Schiff soln. 30 min.; wash. (10) Deacetylation 24 hrs.; wash; periodic acid 15 min.; wash; Schiff soln. 30 min.; wash. (11) Diastase 30 min.; wash; periodic acid 15 min.; wash; Schiff soln. 30 min.; wash. (12) 0-5 per cent, toluidin blue 30 min.; wash. (13) 0-2N NaOH 15 min. at 22° C; wash; 0-5 percent, toluidin blue 30 min. (14) o-2N NaOH 15 min. at 22° C; wash; periodic acid 15 min.; wash; hydroxylamine 60 min.; wash; Schiff soln. 30 min.; wash. (15) Periodic acid 15 min.; wash; hydroxylamine 60 min.; wash; Schiff soln. 30 min.; wash. (16) Periodic acid 15 min.; wash; hydroxylamine 24 hrs.; wash; Schiff soln. 30 min.; wash. (17) Hydroxylamine 1 hr.; wash; o-iN HC1. 15 min.; wash; Schiff soln. 30 min.; wash. (18) Periodic acid 15 min.; wash; Schiff soln. 30 min.; wash in three changes of sulphite soln., 5 min. in each change. 306 Hale—Observations on the Periodic AcidjSchiff Test (19) o-2N NaOH 15 min. at 22° C.; wash; periodic acid 15 min.; wash; Schiff soln. 30 min.; wash in three changes of sulphite soln., 5 min. in each. SIGNIFICANCE OF METHODS AND RESULTS (1) This is the standard technique used to identify . CHOH. CHOH., .CHOH.CHNH2., and .CHOH.CHNHR. groups by production of alde- hydes by periodic acid oxidation and identification of the aldehydes by re- colourization of Schiff's solution. (2) Immersion directly in Schiff's solution, which gives no reaction, shows that none of the colour produced in (1) is caused by the presence of 'free' aldehydes in the sections or by spontaneous recolouration of the Schiffs solution. (3) This is the method which increases the intensity of the staining reaction of certain substances which otherwise give only a pale colour with procedure (1). The maximum amount of change from the standard appearance occurred in the first few minutes of incubation of sections in o-2N NaOH at 220 C. Times or temperatures in excess of this produced no further change. When compared with the results from (1) the sections of human rectum and rabbit duodenum showed the most striking changes. The Brunner's glands of the rabbit duo- denum changed from a faint pink colour with the standard technique to a deep magenta colour after treatment with the NaOH solution (fig. 1, A and B). The goblet cells of the human rectum changed from a pink to a very deep magenta colour (fig. 1, c and D). The fibrin clot of the pneumonic exudate of the lung increased slightly its depth of colour after treatment. In all the tissues there was a slight increase in intensity of staining of reticulin, certain irregular areas of collagen and sarcolemma, and in the amount of diffuse pink connective tissue background. (4) Incubation of sections in NaOH, without subsequent periodic acid oxidation, followed by immersion on Schiff's solution was done to find out if aldehydes were being produced.