Type of presentation: Poster

LS-12-P-3520 Comparing immunolabelling properties in HeLa cells embedded in LR White, Lowicryl, or LR Gold resins after chemical or cryo fixation

Philimonenko A. A.1, Philimonenko V. V.1, Janda P.2, Hozák P.1
1Institute of Molecular Genetics, Prague, Czech Republic, 2J. Heyrovsky Institute of Physical Chemistry, Prague, Czech Republic
tolja@img.cas.cz

Ultrastructural immunolabelling brings important information about localization of biological molecules inside the cell compartments and is an indispensable tool of cell biology (Roth, 1989; Roth et al., 1981; Roth and Taatjes, 1998). For optimal result, it is necessary to preserve well both the fine structure and the antigenic properties of the sample. The choice of optimal technique for the sample preparation is therefore crucial. During the preparation of samples for post-embedding immunolabelling, the antigenic properties of the biological material are influenced by a number of factors, the most important being the fixation method, dehydration procedure, and the resin properties (Skepper, 2000; Stirling, 1990). Classical epoxy resins are in most cases not suitable for subsequent immunolabelling, therefore, a number of acrylic resins have been formulated, which, along with milder fixation, help to overcome the drawbacks of epoxy resins in the antigen preservation and accessibility (Acetarin et al., 1986; Carlemalm et al., 1985).
We compared four acrylic resins - LR White, LR Gold, Lowicryl HM-20, and Lowicryl K4M with regard of immunolabelling efficiency on ultrathin sections. Antigens were detected using standard immunogold technique in either chemically fixed or high-pressure frozen and freeze-substituted HeLa cells after embedment into each of the listed resins. We observed significant differences in immunolabelling densities between studied resins; however, the influence of the resin type was fixation-dependent and antigen-dependent. We recommend using LR White as a standard starting option, keeping in mind that individual optimizing of sample preparation conditions and resin choice may be needed for some antigens. Additionally, the influence of resin surface nanostructure on the immunolabelling efficiency will be discussed.

Acetarin, J.D., E. Carlemalm, and W. Villiger. 1986. J.Microsc. 143:81-88.
Carlemalm, E., W. Villiger, J.A. Hobot, J.D. Acetarin, and E. Kellenberger. 1985. J.Microsc. 140:55-63.
Roth, J. 1989. Methods Cell Biol. 31:513-551.
Roth, J., M. Bendayan, E. Carlemalm, W. Villiger, and M. Garavito. 1981. J Histochem Cytochem. 29:663-671.
Roth, J., and D.J. Taatjes. 1998. Histochem Cell Biol. 109:545-553
Skepper, J.N. 2000. J.Microsc. 199:1-36.
Stirling, J.W. 1990. J Histochem Cytochem. 38:145-157.

This work was supported by the TACR (TE01020118); the project „BIOCEV – Biotechnology and Biomedicine Centre of the Academy of Sciences and Charles University“ (CZ.1.05/1.1.00/02.0109) from the European Regional Development Fund; the IMG institutional grant (RVO68378050).

Fig. 1: Relative labelling densities (LD) of four antigens in the nucleoplasm of HeLa cells after chemical fixation and embedment into Lowicryl HM20, LR Gold or LR White. LD for Lowicryl HM20 was set as 100%. * - P<0.05, ** - P<0.01.