Clawed Frog Nuclear Envelope Protocol

Application Note for Leica EM CPD300 - Life Science Research

October 06, 2016

Introduction:
Species: Clawed frog (Xenopus laevis)
Critical point drying of nuclear pores from clawed frog oocytes with subsequent chromium coating and SEM analysis

Procedure:
Sample Holder: Silicon chips containing the samples were placed into the filter discs and porous pots holder.

Preparation:
Isolated nuclear envelopes were prepared from Xenopus oocytes as described by Goldberg MW, Fiserova J. (2010) Immunogold labelling for scanning electron microscopy. Methods Mol Biol. 657:297-313.


Fixation and Dehydration:

2% Glutaraldehyde, 0.2% Tannic acid, 0.1M Hepes buffer1x10 min.
Distilled water2x1 min.
0.1% aqueous OsO41x10 min.
Distilled water3x10 min.
Ethanol series: 50%, 70%, 95%1x2 min.
Ethanol series: 100%2x2 min.

Leica EM CPD300 auto program:

Coating:
Chromium: 1.5 nm

Fig.1: Nuclear pores from clawed frog oocytes
Fig.2: Nuclear pores from clawed frog oocytes

Comments