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A Valuable Stain for Connective Tissue, Keratin and Fungi* Michel Prunieras, M.D

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A Valuable Stain for Connective Tissue, Keratin and Fungi* Michel Prunieras, M.D View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector PAB: A VALUABLE STAIN FOR CONNECTIVE TISSUE, KERATIN AND FUNGI* MICHEL PRUNIERAS, M.D. Since the papers by Steedman (1), Lison (2)tion. Most of the blocks were freshly prepared and Mowry (3), the use of Alcian Blue stain hasbut some were as old as 30 years. undergone considerable change. The PAB stain (routine) runs as follow: As pointed out by Pearse (4), staining with Alcian Blue is increased when acidic groups are Deparafflo and bring sections to water. introduced by sulfation or by oxidation, due Oxidize in Permanganate for 10 minutes (2.5% to the salt linkage of the dye with acidic groups.MnO4K: 100 cc.; 5% S04112: 100 cc.; distilled water: 700 cc.). The specificity of the stain might also be im- Bleach in 2% oxalic acid, 30 seconds. proved by lowering the pH of the staining bath, Wash in running water and rinse in distilled thus making Alcian Blue staining specific forwater. strong acidic groups, as shown by Adams and Stain ooe slide 30 minutes in 0.1% Alcian Blue Sloper (5). Different oxidative procedures,8GX (Imperial Chemical Industries) in 3% acetic followed by Alcian Blue stain at various pHacid (pH 2.7, 3.0) and one other slide 10 minutes have been already described in the literature:in 1% Alcian Blue in 10% sulphuric acid (pH 0.2, 0.4). A third slide might be stained 1 minute in performie acid (Adams and Sloper, 5), per-1% Alcian Blue in distilled water. manganate (Herlant, 6, Goslar, 7, Aehten, 8). Wash in running water and rinse. Results of such initial oxidation have been re- Counterstain either with hemalin followed by ported in posterior pituitary principles (5),30 seconds in 1% Tueheetgelb (Ciba) or with hypophysis glyeoproteins (6), ringsnake, rat and0.1% I<erneebtrot (Merck) in 5% aluminium human skin keratin (7) and rat and humansulfate. Orcein (Carlo Erba) may be combined, epidermal cell keratinization (8). using 0.10 g. orcein in 70% alcohol: 100 cc. plus This paper deals with preliminary applicationNitric acid: 2 cc., 24 hours. of Permanganate Aleian Blue (PAB) in con- Dehydrate, clear in xylene and mount. nective tissue, keratin and fungi. Routine control slides include: MATERIALS AND METHODS Staining in 0.1% Alcian Blue in 3% Acetic acid. Staining in 0.01% Toluidine Blue (Geigy) in Samples of normal skin were taken from thedistilled water. palms, wrist, chest, abdominal and lumbar Periodic Acid Schiff according to MeManus areas, thighs and legs, armpits, penis, neck and(Schiff reagent prepared according to the scalp. Lougley's modification),(9), combined with Biopsy specimens were taken from 467 patientsAlcian Blue according to Mowry (3). as follows: skin cancers, 345, selerodermas, 38, systemic lupus erythematosus, 12, granuloma The following histochemieal controls were annulare, 25, neerobiosis lipoidica, 10, circum-performed: scribed myxedemas, 5, dermatomyositis, 8 and miscellaneous, 24. For metachromasia, staining 10 minutes in Forty eases of deep myeoses and 48 cultures on0.01% Toluidiue Blue, in buffered solution (Mcllvaiue) at pH 2.2, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0. slides prepared from Micros porurn Gypseum Effect on metachromasia of 0.5%, to 5% barium were also stained with the following technic. and uranium nitrate according to Landsmeer, The slides were cut out of blocks fixed inmodified (10). Bouin's fluid or in Baker's formol-ealeium solu- For basophilia, staining 10 minutes in 0.1% methylene blue (RAL) in buffered solution *Fromthe Clinique Dermatologique, (Direc- tor: Prof. Fr. Woringer) Faculté de médecine, (Mellvaine) at pH 2.2, 4.0, 6.0, 8.0. Strasbourg, Fraaee. Enzymatic digestion with amylase (controlled Received for publication December 30, 1959. saliva test) 30 minutes at 37° C., hyaluronidase 309 310 THEJOURNAL OF JNVESTrGATIVE DERMATOLOGY Fluka) 170 THU/cc., four hours at 37° C. in saline), these cells is readily obtained by lowering the pH pyocyanine (Hoffmann-LaRoche) 0.1% in distilled of the dyeing solution. water (pH 6.8), five hours at 37° C., according to That Aleian Blue alone stains only part of the Lefebvre (11). tissue mast cells is demonstrated by the following The slides were acetylated and saponified ac- experiment: cording to McManus and Cason; pyridinc (Merck) extracted or methylated according to Pearse (4). One slide is stained with Alcian Blue acetic, Oxidations with 2.5% permanganate in distilledwithout oxidation and then counterstained with water were performed for various times at roomToluidine Blue at various pH. Metachromasia, temperature. Combined permanganate oxidationspecific for mast cells is easily recognized at the and periodic oxidation was tested according tolevel of many tissular elements that are Aleian Achten (8). Blue negative. In certain pathological eases, Sulfation was experienced by the sulfuric acid-such as circumscribed Myxcdema, one can find ether technic according to Mowry, quoted bynot only metaehromatic cells that are Alcian Herlant (6). Blue negative, but also Alcian Blue positive cells The effect of oxidative solutions was tested as follows: one test slide is stained with Toluidinecontaining one or two clots of metachromatie Blue and metachromasia is checked first, imme-material located near the nucleus. diately in water and second, after blot-and-air Mature, trisulfated heparin containing mast dehydration. Then the coverslip is removed, thecells do react directly with Alcian Blue as is slide is oxidized, restained and rechecked. Finally,shown by the still positive metaehromasia in tbe slide is decolorized through alcohols andthese cells at such low pH as 2.2. Others do not. stained with Alcian Blue. These (immature) cells are Schiff-positive after periodic oxidation. They, also, are Alcian Blue RESULTS positive after permanganate oxidation. Great variations occur according to the type Hence, oxidation with permanganate hefore of fixation. After Bouin's fluid fixation, whichAlcian Blue staining reinforces the colorability of immature mast cells, as compared with Alcian contains picric acid, direct staining with Alcian Blue is poor or negative except for sulfuricBlue alone. solution and for the dermal papilla of growing On the other hand, as compared with Toluidine hairs, which is positive both with acetic andBlue metaehromasia, it is demonstrable that oxidation with sulfuric permanganate increases sulfuric solutions. the number of metachromatic cells. After Baker's formol-ealcium fixation, staining with Alcian Blue, either acetic or sulfuric re- Such an increase depends upon the type of tissue, the site of biopsy and the fixation fluid. produces what has already been extensivelyIt may vary along a rather wide range, i.e. from reported: Cawley, MeManus, Lupton andalmost twice as many to nothing. Wheeler, (12, 13), Goltz, Fusaro and Jarvis, Nevertheless, as contingent as that difference (14), Braun-Faleo, (15, 16, 17), Steigleder, (18). Oxidation with permanganate induces twocould be, the fact remains that after Bouin's fixation or formol-calcium fluid, one cannot, at main changes: Certain structures become positive that wereall times, demonstrate with Toluidine Blue alone, all the cells containing metaehromatic negative before oxidation. granules in dermal connective tissue. It must be Certain structures become negative that were emphasized that Aleian Blue in sulfuric solution positive before oxidation. The most intense positive reactions are ob-after oxidation (PABS) precisely stains as many tained with mast cells, keratin and fungi. Othermast cells as does Toluidine Blue after the same structures offer less demonstrative results. oxidation. As for tissue mast cells, one obtains two com- Among other structures that become positive plementary effects: bright blue-green stainingafter oxidation, keratin is one of the most re- and increased number of reacting cells. active. Without oxidation, the number of mast cells Hard keratin, soft keratin type Zander A or revealed by Alcian Blue is scarce and con-Zander B are negative without oxidation. After tingent. A definite increase of the reaction inoxidation, the type Zandcr A reacts with PABS PAB: STAIN FOR TISSUE, KERATIN AND FUNGI 311 in the layers immediately above the granulosa. Among cells, no other cell than mast cells react This positivity decreases as one goes up to thewith PABS. With PABA, on the contrary, more superficial layers. ordinary basophilie cytoplasms react more or With PABA, the reaction is noticeably strongerless. Plasma cells deserve particular notice in and persists till the highest portions of the hornythis respect. They exhibit blue color in their layer. There may be found some reactive materialeytoplasma distinct from the green hue of the in the cytoplasm of some scarce cells near themast cells eytoplasms. Also is noticeable that de- granulosa. Methylation suppresses the PABAfinite positive granules may be seen in fibroblasts. reaction. Aeetylation does not, except for that At the level of extracellular connective tissue, cytoplasmie material quoted above. elastic tissue reticulin fibers, basement mem- The increase in positivity when one turns frombranes and cementing substances do stain either PABS to PABA corresponds to the PAS posi-in normal or in pathologic skin. Considerable tivity and to metaehromasia possibly induced bychanges occur according to the type of fixation permanganie oxidation. The reactivity to PABSon one hand and to the type of tissue on the is not affected by prior extraction with pyridine.other. Zander B type heratin grossly reacts in the What must be emphasized, however, is the same way. PABS may be negative. Hard keratinpositive PABA reaction of glycogen and glyeo- of hairs is strongly reactive as well with PABSprotein complexes. as with PABA. As far as glycogen is concerned, one obtains The inner root sheath is strongly positivePABA positive reaction whereas PABS remains after oxidation. However, one observes at timesnegative. Partial loss of reactive material occurs faint Alcian Blue staining in the Huxley layerunless the slides are coated with a eollodion-film before oxidation.
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