Placental Alkaline Phosphatase Labeling for Tracing Transgenic Mouse Retinal Bipolar Cells in 3D Serial Blockface Scanning Electron Microscopy Datasets

Abstract

Reconstructing the 3D organization of individual neurons and their processes within the complex cyto-architecture of the nervous system is important for unraveling key neuronal pathways. Recent advances in serial block face (SBF) scanning electron microscopy (SEM) technologies have made acquisition of large 3D datasets of serial EM "sections" a comparatively routine technique. Both dual beam instruments and in-chamber ultramicrotome approaches (Gatan 3View) utilize backscattered electrons generated by heavy metal impregnation of tissue elements in the sample. Sample contrast is a key limitation for image acquisition and analysis. For many SBF experiments, identifying individual cells of interest is critical, and genetic approaches promoting heavy metal labeling of cells and organelles have been described.These include photoconversion of DAB by enhanced green fluorescent protein [1] or by singlet oxygen generated by miniSOG constructs [2]. Transgenic expression of placental alkaline phosphatase (PLAP) offers potentially a very useful means for labeling cells. A wide range of substrates make it useful for fluorescence and brightfield light microscopy, and PLAP has also been used in EM as a transgenic cell marker [3]. It is well suited as a reporter for cell identification as PLAP localizes to the outer surface of the plasma membrane and so reaction products do not obscure internal ultrastructural detail. While successfully applied to TEM, serial blockface imaging requires substantially higher heavy metal staining densities than TEM. In this study we investigated the suitability of PLAP labeling, in combination with heavy metal counterstaining, for cell reconstruction by 3D SBF-SEM.Transgenic mice were obtained in which the human placental alkaline phosphatase (PLAP) is selectively synthesized by retina bipolar cells expressing express the homeobox gene, Chx10. To permit EM tracing of adult mouse retinal cells, mice were perfused with 2% paraformaldehyde and 1% glutaraldehyde 0.1M phosphate buffer after an Ames buffer pre rinse. PLAP reactivity was detected by en bloc incubation in a -glycerophosphate, alkaline lead citrate according to Mayahara [4]. A Leica tissue slicer was used to cut 100 µm thick slices and these specimens were prepared as described by Gustinich [5]. Briefly the tissue was fixed an additional hour in the perfusion fixative 160