High-contrast en bloc staining of neuronal tissue for field emission scanning electron microscopy (original) (raw)

Conventional heavy metal post staining methods on thin sections lend contrast but often cause contamination. To avoid this problem, we tested several en bloc staining techniques to contrast tissue in serial sections mounted on solid substrates for examination by Field Emission Scanning Electron Microscope (FESEM). Because FESEM section imaging requires that specimens have higher contrast and greater electrical conductivity than transmission electron microscope (TEM) samples, our technique utilizes osmium impregnation (OTO) to make the samples conductive while heavily staining membranes for segmentation studies. Combining this step with other classic heavy metal en bloc stains including uranyl acetate, lead aspartate, copper sulfate and lead citrate produced clean, highly contrasted TEM and SEM samples of insect, fish, and mammalian nervous system. This protocol takes 7-15 days to prepare resin embedded tissue, cut sections and produce serial section images. The authors of this manuscript declare that they have no competing financial interests. Author contributions JB developed the staining concept, prepared the fish and Drosophila samples and prepared the manuscript. JCT, NK, and RS imaged the Drosophila sections. RS assisted with the sectioning, imaging and overall block quality assessment. KH, RS, JCT and NK improved ultra thin sectioning and collection. KH developed the method of collecting ultrathin sections on tape and built the ATUM devices used. JWL helped motivate the effort to find better en bloc staining protocols, oversaw all the imaging experiments that were carried out in his laboratory and helped interpret the image data. SJS helped motivate the effort to improve en bloc staining, and oversaw and assisted with imaging experiments carried out at Stanford.

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