Use of periodate-lysine-paraformaldehyde for the fixation of multiple antigens in human skin biopsies

Abstract

Periodate-lysine-paraformaldehyde (PLP) has been proposed as a fixative for glycoprotein antigens which should stabilize periodate oxidized polysaccharide chains through lysine mediated crosslinks, either directly or by the intermediation of formaldehyde. In spite of premises and attempts reported in the literature, this fixative has never become popular for the study of membrane antigens of immune system cells, which leads to doubts on its real efficacy. We have addressed this issue in biopsies of human skin and found that PLP followed by cryoprotection with 30% sucrose and cryosectioning, or PLP fixation of isolated epidermal sheets, consistently provided for good preservation of morphology and intense labeling of major histocompatibility complex class II molecules, CD1a, CD4, CD8, E-cadherin, cytokeratins in general, cytokeratin-18 in particular, and bromodeoxyuridine, incorporated by cycling cells in vitro, and for the demonstration of tyrosinase enzyme activity. PLP-fixed, osmicated and epon-embedded epidermal sheets proved as good as sheets fixed 365 with a mixture of formaldehyde and glutaraldehyde for electron microscopic morphological analysis. Also, these sheets were amenable to immunoperoxidase staining of Langerhans cell membrane antigen CD1a and keratinocyte membrane antigen E-cadherin before being osmicated and prepared for electron microscopy. In a parallel paper, we had also shown that oral mucosa biopsies fixed in PLP showed good morphology and immunolabeling of CD54, CD80, CD83 and CD86. Therefore, we conclude that PLP can be proposed as a multi-task fixative for light and electron microscopic analysis of membrane, cytoplasmic and nuclear antigens of immune system cells and keratinocytes.

Keywords

• bromo-deoxyuridine
• cryosections
• cytokeratins
• Langerhans cells
• membrane antigens
• PLP
• tyrosinase