Comparison of methods of high-pressure freezing and automated freeze-substitution of suspension cells combined with LR White embedding

Abstract

In this study we present an optimized method of high-pressure freezing and automated freeze-substitution of cultured human cells, followed by LR White embedding, for subsequent immunolabeling. Also, the influence of various conditions of the freeze-substitution procedures such as temperature, duration, and additives in the substitution medium on the preservation of cryo-immobilized cells was analyzed. The recommended approach combines (1) automated freeze-substitution for high reproducibility and minimizing human-derived errors; (2) minimal addition of contrasting and fixing agents; (3) easy-to-use LR White resin for embedment; (4) good preservation of nuclei and nucleoli which are usually the most difficult structures to effectively vitrify and saturate in a resin; and (5) preservation of antigens for sensitive immunogold labeling.

Quantitative evaluation of freeze-substitution effects on preservation of nuclear antigens during preparation of biological samples for immunoelectron microscopy

“…The cell pellets were then high-pressure frozen in the Leica EM PACT2 (Leica Microsystems AG, Wetzlar, Germany) high-pressure freezer as discussed previously (Sobol et al 2010), and the frozen samples were freezesubstituted in the automatic FS machine Leica EM AFS2 equipped with an automatic reagent handling system Leica EM FSP. The FS methods in the automatic FSP mode of the Leica EM AFS2 machine were as described previously (Sobol et al 2010).…”

“…The cell pellets were then high-pressure frozen in the Leica EM PACT2 (Leica Microsystems AG, Wetzlar, Germany) high-pressure freezer as discussed previously (Sobol et al 2010), and the frozen samples were freezesubstituted in the automatic FS machine Leica EM AFS2 equipped with an automatic reagent handling system Leica EM FSP. The FS methods in the automatic FSP mode of the Leica EM AFS2 machine were as described previously (Sobol et al 2010). Briefly, in Method A, the cells were freeze-substituted in acetone (Lach-Ner s.r.o., Neratovice, Czech Republic; dehydrated using molecular sieves, Sigma-Aldrich) from -90°C to 0°C for 111 h followed by LR White resin (Sigma-Aldrich) embedment (0°C, 50 h) and polymerization (4°C, 128 h).…”

“…It is necessary to mention here that cryoimmobilized and freeze-substituted cells are embedded into acrylic resins which are better suited for immunocytochemical studies (Acetarin et al 1986;Roth 1989;Roth and Taatjes 1998;Roth et al 1981). Among them, Lowicryl resins are usually chosen, but LR White is the resin of our first choice that was previously discussed (Sobol et al 2010;Sobol et al 2011). …”

Histochemistry and cell biology: the annual review 2010

“…Future studies will therefore focus on MR imaging, biodistribution and pharmacokinetics of these particles in animal models. Sobol et al (2010) aimed to optimize the procedure of cryoimmobilization of suspension cells for electron microscopy. The authors therefore examined various conditions of freeze-substitution procedures and determined conditions that allowed high reproducibility and most importantly good preservation in particular nuclei and nucleoli (considering the fact, that such intracellular target sites represent the most diYcult structures with regard to eVective veriWcation and saturation with resin), in addition to antigens for immunogold labelling.…”

Closer to the native state. Critical evaluation of cryo-techniques for Transmission Electron Microscopy: preparation of biological samples

“…It has been well documented that cryo-fixation by HPF combined with FS and resin embedding can significantly improve the preservation of ultrastructure and antigenicity [123,124]. Nevertheless, not all antigens can be immunolabelled in such a way due to a limited number of accessible antigens at the resin section surface (especially important in locating rare antigens) [125].…”