TECHNICAL NOTE |
Correspondence to: Robert Stern, Dept. of Pathology, HSW-501, School of Medicine, UCSF, San Francisco, CA 94143-0506.
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Summary |
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The apparent intensity of hyaluronan (HA) staining in tissue sections can vary as a function of fixation techniques. We examined the histochemical distribution of HA in normal human skin using an HA-specific binding peptide derived from bovine nasal cartilage. The HA, particularly in the dermis, was best preserved in sections fixed in 10% acid-formalin with 70% ethanol. In contrast, sections fixed in the routine 10% neutral-buffered formalin had a much weaker intensity of HA staining. Furthermore, acid-formalin/ethanol-fixed sections retained much of their apparent HA after incubation with saline, in contrast to the neutral formalin-fixed sections, in which most of the stainable HA was lost. Such marked differences in staining intensity were not observed in slides stained with Alcian blue, a procedure pressumed to stain HA as well as other glycosaminoglycans. Staining using the HA binding peptide was entirely absent when sections were first preincubated in hyaluronidase, whereas similar Alcian blue-stained sections retained most of their staining intensity. Caution should be exercised in evaluating the distribution of HA in tissues using the HA binding peptide, particularly when different fixation techniques among several laboratories are being compared. In addition, the ability to evaluate the HA content of tissues using Alcian blue staining should be reconsidered. The sulfated glycosaminolglycans of the "ground substance" appear to be the predominant substrates for Alcian blue. (J Histochem Cytochem 45:1157-1163, 1997)
Key Words: hyaluronan, glycosaminoglycans, fixation, human skin, Alcian blue
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Introduction |
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More than 50% of total body hyaluronan (HA) is present in skin (
Recently, a technique for the histolocalization of HA (
The distribution of HA in human skin has been described using the HABP technique (
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Materials and Methods |
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Tissue Samples
The sample of human skin, obtained from a routine autopsy in the Department of Pathology, University of California, San Francisco, was taken from the scalp of a 35-year-old white man. The specimen was divided in two and one sample was fixed in 10% formalin in 70% ethanol and 5% glacial acetic acid (acid-formalin/EtOH), all v/v. The precise formulation of this solution is as follows. For each 100 ml of fixative, 10 ml of stock 37-40% formaldehyde, 70 ml of 100% ethanol, 5 ml of glacial acetic acid, and 15 ml of water were utilized. The other sample was fixed in 10% neutral-buffered formalin, using 0.1 M PBS, pH 7.35, as described (
Histochemical Staining for HA
The HA-specific HABP was prepared as described (
Alcian Blue Staining
The Alcian blue staining was performed in pH 2.5 buffers using a method as described (
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Results |
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In this study, HA staining in skin was found predominantly in the dermis, rather than in the epidermis, particularly in the papillary dermis (Figure 1). The most intense staining was observed in sections fixed with acid-formalin/ethanol (Figure 1A) compared to sections fixed with conventional neutral-buffered formalin (Figure 1B). Of particular interest is that small scattered foci of staining in the epidermal layer were comparable to the intensity of staining found in the dermis using acid-formalin/ethanol (Figure 1A). Such foci in the epidermal layer stained less intensely in conventionally fixed samples (Figure 1B). The staining for HA was blocked by preincubation of the HABP with HA. In addition, preincubation of the HABP with other GAGs such as chondroitin sulfate, dermatan sulfate, and keratan sulfate at the same concentration did not decrease the intensity of subsequent HA staining (data not shown). These results demonstrated that the HABP staining reaction was highly specific for HA and did not react with other tissue GAGs.
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To confirm further that the HA in skin fixed in acid-formalin/ethanol was better preserved than in neutral-buffered formalin, one set of slides was incubated with PBS overnight before the staining reaction. As shown in Figure 2, approximately 80-90% of HA was retained in slides fixed in acid-formalin/ethanol (Figure 2A) compared to sections fixed in neutral-buffered formalin. Notably, the staining of the epidermis was almost unchanged. However, much of the stainable HA in the dermis had leeched out of the sample fixed with neutral formalin during the overnight incubation in PBS (Figure 2B). Therefore, tissue fixed in acid-formalin/ethanol retained HA far better than that fixed in neutral-buffered formalin.
An artifact became evident in the course of these studies. In the acid-formalin/ethanol-fixed sections (Figure 1A), HA staining was most prominent in the upper spinous and granular layers of the epidermis. In the conventional formalin-fixed section, however (Figure 1B), the converse was true. The basal and spinous layers of the epithelium had the most pronounced HA staining, with little staining observed in the outer granular layers. A plausible interpretation of this apparent inversion artifact may be the following. The HA moieties of the outer spinous and granular layers are more loosely associated, whereas the HA of the basal and inferior portions of the spinous layer are more firmly tethered to tissue structures. The aqueous formalin solution is able to dissolve and elute out the HA of the outer epidermal layers, whereas the HA associated with inferior layers of the epidermis are less soluble and thus more resistant to being leeched out.
Alcian blue, a classical histochemical cationic dye, is reputed to stain HA as well as other acidic GAGs. Staining of both HA and the sulfated GAGs is assumed to occur at pH 2.5, whereas preferential staining of the sulfated GAGs occurs at pH 0.5 to 1.0. When sections were stained with Alcian blue at pH 2.5, slightly more intense staining occurred in sections fixed with the acid-formalin/ethanol (Figure 3A) compared to sections fixed in the standard neutral-buffered formalin (Figure 3B). Preincubation with PBS did not cause an appreciable decrease in the intensity of staining in sections using either fixative (Figure 4A and Figure 4B). This suggests that Alcian blue actually stains predominantly the sulfated GAGs and does not stain HA appreciably in the presence of the tissue-fixed sulfated GAGs.
This phenomenon was investigated further. Sections were preincubated with Streptomyces hyaluronidase and the staining was then compared. In both acid-formalin/alcohol- and formalin-fixed sections (Figure 5A and Figure 5C), all staining disappeared after hyaluronidase predigestion (Figure 5B and Figure 5D). However, with Alcian blue staining (Figure 6A and Figure 6C), only a slight decrease in staining intensity was observed after hyaluronidase predigestion (Figure 6B and Figure 6D).
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Finally, we examined for possible artifacts introduced with acid-formalin/alcohol fixation using conventional H&E staining. As shown in Figure 7A, the collagenous fibers of the dermis appear contracted, leaving spaces between fibers, compared to the dermal collagen in the formalin-fixed section (Figure 7B). However, structural preservation and cellular detail otherwise appear comparable between the two methods of fixation.
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Discussion |
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Our results demonstrate that fixation modulates the apparent distribution of HA in human skin. It appears that the acid-formalin/ethanol preserves HA in skin far better than the neutral formalin in PBS and may generally be the fixative of choice for the evaluation of HA in tissues. Formalin crosslinks amino groups of proteins to form methylene bridges. Hyaluronan does not contain such free amino groups but does have - NHCH3 moieties. We have observed that acid-formalin/ethanol solidifies a 1% HA solution, generating a semisolid gel (unpublished observation). This phenomenon was not observed with either 70% ethanol or 5% acetic acid alone or with the neutral-buffered formalin. Furthermore, the semisolid gel remained insoluble during overnight incubation in PBS. Although it is unlikely that formalin crosslinks HA, in the presence of 70% ethanol and 5% acetic acid the formalin appears to modify the structure of HA so that it binds to tissue components in a noncovalent manner. This aggregation or binding is relatively water-insoluble.
Most other GAGs in the ECM are covalently bound to structural proteins to form proteoglycans. In contrast, HA is generally not covalently bound to protein, although exceptions have been described (
Hyluronan and its receptors are taking on increasing importance in biology (Laurent and Fraser 1992), being involved in such basic processes as carcinogenesis (
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Acknowledgments |
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Supported by Hyal Pharmaceutical Corp., Ontario, Canada, and by a DHHS grant from the US National Institutes of Health (GM46765).
Received for publication October 23, 1996; accepted March 10, 1997.
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