VIS2FIX: a high-speed fixation method for immuno-electron microscopy |
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Authors: | Karreman Matthia A van Donselaar Elly G Gerritsen Hans C Verrips C Theo Verkleij Arie J |
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Affiliation: | Molecular Biophysics, Department of Physics and Astronomy, Utrecht University, Princetonplein 1, NL-3508 TA Utrecht, The Netherlands. M.A.Karreman@uu.nl |
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Abstract: | Immuno-transmission electron microscopy (TEM) is the technique of choice for high-resolution localization of proteins in fixed specimen. Here we introduce 2 novel methods for the fixation of sections from cryo-immobilized samples that result in excellent ultrastructural preservation. These high-speed fixation techniques, both called VIS2FIX, allow for a reduction in sample preparation time from at least 1 week to only 8 h. The methods were validated in immuno-TEM experiments on THP-1 monocytes, human umbilical vein endothelial cells (HUVECs) and Madin-Darby canine kidney (MDCK-II) cells. The fixation and retention of neutral lipids is demonstrated, offering unique prospects for the application of immuno-TEM in the lipidomics field. Furthermore, the VIS2FIX methods were successfully employed in correlative fluorescence and electron microscopy. |
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Keywords: | correlative microscopy high‐pressure freezing HUVEC iLEM immuno‐transmission electron microscopy lipid MDCK‐II section fixation THP‐1 VIS2FIX |
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