Cryosectioning and immunolabeling.
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.
Slot
Jan W
JW
Cell Microscopy Center of the Department of Cell Biology, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands.
Geuze
Hans J
HJ
D000818:Animals; D002478:Cells, Cultured; D016616:Cryoultramicrotomy / Q000379:methods*; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D008055:Lipids / Q000032:analysis; D011506:Proteins / Q000032:analysis; D051381:Rats; D016707:Tissue Fixation
D008055:Lipids; D011506:Proteins