A protocol for isolating Xenopus oocyte nuclear envelope for visualization and characterization by scanning electron microscopy (SEM) or transmission electron microscopy (TEM).
This protocol details methods for the isolation of oocyte nuclear envelopes (NEs) from the African clawed toad Xenopus laevis, immunogold labeling of component proteins and subsequent visualization by field-emission scanning electron microscopy (FESEM) and transmission electron microscopy (TEM). This procedure involves the initial removal of the ovaries from mature female X. laevis, the dissection of individual oocytes, then the manual isolation of the giant nucleus and subsequent preparation for high-resolution visualization. Unlike light microscopy, and its derivative technologies, electron microscopy enables 3-5 nm resolution of nuclear structures, thereby giving unrivalled opportunities for investigation and immunological characterization in situ of nuclear structures and their structural associations. There are a number of stages where samples can be stored, although we recommend that this protocol take no longer than 2 d. Samples processed for FESEM can be stored for weeks under vacuum, allowing considerable time for image acquisition.
Allen
T D
TD
Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Withington, Manchester M20 4BX, UK. tallen@picr.man.ac.uk
Rutherford
S A
SA
Murray
S
S
Sanderson
H S
HS
Gardiner
F
F
Kiseleva
E
E
Goldberg
M W
MW
Drummond
S P
SP
D000818:Animals; D002458:Cell Fractionation / Q000379:methods*; D004210:Dissection / Q000379:methods; D007150:Immunohistochemistry / Q000379:methods; D008854:Microscopy, Electron / Q000379:methods; D009685:Nuclear Envelope / Q000648:ultrastructure*; D009865:Oocytes / Q000648:ultrastructure*; D014982:Xenopus laevis