Okajimas Folia Anat. Jpn., 84(1): 11–18, May, 2007 Significance of PAM Histochemical Reaction in Delineating Macrophages By Yuji SHINYA1,KazuyaHAMADA1,P.D.GUPTA2 and Fumioki YASUZUMI1 1Department of Physiological Science Anatomy 2. Faculty of Medicine, University of the Ryukyus, 207 Uehara, Nishihara, Okinawa, 903-0215 Japan 2Atmiya Institute of Gerontology Research, Yogidham, Kalawad Road, Rajikot-360 005, Gujarat, India – Received for Publication, December 29, 2006 – Key Words: Macrophage, Fetal macrophage, Placental villi, Periodic acid methenamine silver, Glycol-methacrylate Summary: Periodic acid methenamine silver staining has been used for proof of the existence of carbohydrates in both paraffin and resin embedded tissues. The staining was applied to delineate macrophages that have been well known as cells of mononuclear phagocyte system by stainable nature for lysosomes, and it is found to be useful method for de- tection of macrophage. Fetal macrophage, which is often observed in placental villi, is called the highly vacuolated cell. Vacuoles in the cytoplasm seem to be empty with light microscopic staining, but it is clarified that they contain a full amount of carbohydrate. The present result leads us to the necessity of reconsidering the functional aspect of fetal macrophage. Periodic acid Schiff (PAS) reaction is specific In the present study, without using enzyme his- histochemical staining for carbohydrates and was tochemistry in tissues embedded in GMA by PAM introduced by McManus (1948)1).Periodicacid staining we have delineated lysosomes in various methenamine silver (PAM) is a modified method of types of macrophages including fetal macrophages PAS reaction; the superiority of PAM over the which were not identified by this staining method so PAS was seen in glomerular basement membrane. far. Histochemically, positive objects in PAM reaction having very high contrast compare to the sur- rounding tissue components2). Rambourg and Leb- Materials and Method lond used this staining method directly on epoxy resin section, and examined specificities of the Fixation and embedding staining by taking proper controls i.e., staining the All tissues collected from various animals and tissue where periodic acid was ommited3).Intheir tissues, mentioned below were fixed for 12 hours in report one of the cell organelles, the lysosomes 2.5% glutaraldehyde adjusted to pH 7.4 with were stained by PAM staining method. The lyso- 0.05 M cacodylate buffer, and then washed with the somes also stained positive in PAM4) staining same buffer and dehydrated routinely with alcohol, method when glycol methacrylate (GMA) em- and embedded in GMA (Technovit 7100, Kulzer). bedded sections is used. Since proposed the mononuclear phagocytic sys- Kupffer cells (liver macrophages); Wister rat liver. tem (MPS) consisting of various types of macro- Intestinal macrophages from lamina propria; phages such as, histiocyte, Kupffer cell, osteoclast, monkey rectum. Langerhans cell, Hofbauer cell, etc5,6),arethemain Testicular macrophages from human (testis was defense cells in the body5). The macrophages have removed from prostate cancer patient). many lysosomes which show acid phosphatase Fetal macrophages (Hofbauer cells), from the activity as a marker enzyme5–7). human placenta of eight week gestation. Yuji Shinya, Department of Physiological Science Anatomy 2. Faculty of Medicine, University of the Ryukyus, 207 Uehara, Nishi- hara, Okinawa, 903-0215 Japan. 11 12 Y. Shinya et al. The specimen were sectioned at 4 mm thickness on the main cells for the defense system of the body, Sorvall JB-4 microtome fitted with Ralph’s glass called the mononuclear phagocyte system. One of knife8). Every section was re-sectioned from tissue the characteristic feature of these cells is that each blocks embedded in GMA for histological practices. cell contains a number of lysosomes5) in the cyto- plasm. Acid phosphatase activity, the maker en- Staining zyme11) of the lysosomes is also very high in these HE staining cells. 1) Carazzi’s hematoxylin9) modified staining10): In 1967 Rambourg and Leblond used PAM Sections were stained with the modified solution for staining at ultrastructure level and were able to 24 hours. stain mucin, basement membrane, lysosomes, and 2) Eosin staining was followed as described ear- Golgi saccules however, when the section was not lier4). Toluidine blue10) was used for semi-thin resin subjected to periodic acid, lysosome was not stained sections. with methenamine silver, but nuclei and some pig- PAM staining and PAM with gold chloride ments were stained non-specifically even without method was followed according to Jones2). Control periodic acid treatment3). sections were not treated with periodic acid prior to treatment with methenamine silver3). Kupffer cells Photographs of the same section were taken Kupffer cells are located in the liver sinusoid, after different staining as described by Nakamura attached to the endothelial cells and contain many and Yasuzumi4). Olympus light microscope (BX50) black granules in the cytoplasm that are stained fitted with digital camera (PDMC Ie/OL) was used with PAM. These granules are homologous with for photography. granules of phagosomes6), that is, secondary lyso- somes (acid phosphatase positive)11) (Figs. 1A, 1B). Results and Discussion Intestinal macrophages There are many kinds of mesenchymal cells, such Since the report of van Furth et al.5) had been as fibroblast, macrophages, lymphocytes, plasma published, the macrophages has been recognized as cells, neutrophils, eosinophils, mast cells and adi- Fig. 1. Kupffer cell of the rat liver. HC: liver parenchymal cell, K: Kupffer cell, S: sinusoid. A. Kupffer cell stained with PAM. In the cytoplasm of the cell, dense granules considered secondary lysosomes are stained with PAM specifically. Both nuclei of liver parenchymal cell and Kupffer cell are stained non-specifically. Lysosomes of paren- chymal cell are stained palely (short arrow). Â1,350. B. Same field of A is stained with additional HE staining. Liver structures in the lobules is clarified increasingly by HE staining. Adjusted to liver cell cord the sinusoid, in which condensed erythrocytes and the Kupffer cell locates in the lumen, is clearly observed. Â1,350. PAM Staining in Delineating Macrophages 13 Fig. 2. Intestinal macrophages in the monkey rectum. The same section is stained with different ways of staining. e: eosinophil, f: fibroblast, ly: lymphocyte, m: macrophage, ms: mast cell, p: plasma cell. A. A section which is stained with toluidine blue differentiates mast cell by metachromasia, plasma cell by basophilic cytoplasm, and macrophage by blueish lysosomes. Â830. B. The section of A is stained with HE after A. HE stains the granules of eosinophil positively. Â830. C. The section of B is stained with PAM after B. PAM stains lysosomes black. Â830. pose cells in the lamina propria of the intenstine12). somes6) and in fact contain acid phosphatase, but Especially macrophages aggregated on the tips of PAM does not stain them (Figs. 2A, 2B, 2C). the intestinal villi and arranged at the base the epi- thelium were proved by means of acid phosphatase Testicular macrophages activity of lysosome13,14). When a GMA section of The seminiferous tubule which destines to be the rectal mucosa is stained with different ways of collapsed by ageing process undergoes atrophy, staining successively, toluidine blue, HE, and then thickening of the basement membrane, and dis- PAM, in the same visual field4), the mast cell is dif- appearing of all epithelial cells finally from the ferentiated with toluidine blue from other cells by tubule16). In the process of collapsing, the macro- metachromasia (Fig. 2A). Macrophages in the lam- phages impinge the basement membrane, enter the ina propria are in larger number in which lyso- tubule and phagocyte degenerated cells and cell somes are stained basophilic and slightly bluish debris17). In the cytoplasm of the macrophage, a with toluidine blue, faint eosinophilic with HE, and number of condensed lysosome are detected by black intensely with PAM. The eosinophil is de- PAM stain (Fig. 3A). With HE staining, normal tected by staining nature of specific granules which spermatogenic cell is well demarcated (Fig. 3B). A are stained negatively with toluidine blue but eo- completely collapsed residual tubule show thick- sinophilic with HE15). Nevertheless specific granules ened basement and luminal membranes, which is of eosinophil have been considered one of the ly- considered to be the surface of the lamina lucida of sosomes6) and in fact contain acid phosphatase, the basement membrane (Figs. 3C, 3D). PAM does not stain then (Figs. 2A, 2B, 2C). Therefore, lysosomes, specific granules of the eo- Fetal macrophages or Hofbauer cells sinophil, are thought to be primary lysosomes. The Hofbauer cells are one of the component of eosinophil is detected by staining nature of specific mononuclear phagocytes system17), which is based granules which are stained negatively with toluidine on the mesenchymal stromal cell of placenta and blue but eosinophilic with HE15). However, they fetus. These cells are highly vacuolated cells. The are not stained with PAM. Specific granules of eo- vacuoles show positive reaction to acid phosphatase sinophil have been considered one of the lyso- activity18). The cells are more prominent in the first 14 Y. Shinya et al. Fig. 3. Testicular macrophage. B: basement membrane, m: macrophage, SL: Sertoli cell, SP: spermatogenic cell. A. A section is stained with PAM. Testicular