Abstract
In this protocol, we describe cryoimmunolabeling methods for the subcellular localization of proteins and certain lipids. The methods start with chemical fixation of cells and tissue in formaldehyde (FA) and/or glutaraldehyde (GA), sometimes supplemented with acrolein. Cell and tissue blocks are then immersed in 2.3 M sucrose before freezing in liquid nitrogen. Thin cryosections, cut in an ultracryotome, can be single- or multiple immunolabeled with differently sized gold particles, contrasted and viewed in an electron microscope. Semi-thin cryosections can be used for immunofluorescence microscopy. We describe the detailed procedures that have been developed and tested in practice in our laboratory during the past decades.
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Acknowledgements
We are very grateful for the long and excellent collaboration with Elly van Donselaar, Janice Griffith, Viola Oorschot and George Posthuma. Their technical skills and devotion to deliver optimal results were essential to set the standards of the above protocols. From outside, our group we like to acknowledge Dr. Gareth Griffiths with whom we had fruitful discussions all over the years. In particular, we thank Dr. Kyoteru Tokuyasu, our master.
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Slot, J., Geuze, H. Cryosectioning and immunolabeling. Nat Protoc 2, 2480–2491 (2007). https://doi.org/10.1038/nprot.2007.365
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DOI: https://doi.org/10.1038/nprot.2007.365
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