A method for preserving ultrastructural properties of mitotic cells for subsequent immunogold labeling using low-temperature embedding in LR White resin

Abstract

The best available approach of biological sample preparation for transmission electron microscopy currently includes cryoimmobilization by high-pressure freezing (HPF) followed by freeze-substitution (FS). This method has been well established for interphase cells; however, a reliable and easy procedure is still missing for mitotic cells especially because of their fragility and sensitivity to treatments. Here, we present a fast and effective method for HPF/automated FS and LR White embedding of mitotic cells which allows for their controlled and reproducible quality processing. It should be useful in various ultrastructural studies on mitotic cells especially in combination with immunocytochemistry.

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

“…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). …”

Recent progress in histochemistry and cell biology

“…The cell nucleus in general is the most sensitive organelle for cryofixation (Buser and Walther 2008;Wild et al 2001) and chromatin one of the most difficult structures to vitrify (Martens et al 2009). Therefore, focusing on mitotic cells, Sobol et al (2011) successfully set out to optimize the approach of HPF/automated FS and LR White embedding of mitotic cells in order to optimize preservation of the ultrastructure and antigenicity, i.e., to make samples serviceable to subsequent immunolabeling protocols.…”

Avaliação histológica e histomorfométrica da energia superficial e molhabilidade em implantes de titânio grau IV e grau V: estudo experimental em coelhos

“…Com a conclusão das fases cirúrgicas, as amostras coletadas ficam submersas em formol a 10% até o início dos procedimentos de desidratação das amostras e inclusão em resina, conforme seus protocolos (SOARES et al, 2015;PIATELLI et al, 2012;SOBOL et al, 2011).…”

“…Para isso, a resina deve possuir boas propriedades de infiltração com impregnação dentro das amostras, com baixa viscosidade e polimerização em baixas temperaturas, além de ser hidrofóbica após polimerização. A resina acrílica pode ser da marca LR WhiteResin Medium Grade (London Resin Company, Berkshire, UK), utilizada nesta pesquisa e em pesquisas com animais pelos seguintes autores, Soares et al, 2015;Sobol et al, 2011;Oliveira et al, 2009 Sartoretto et al, 2015;Stancari, 2015e 2011. Segundo Soares et al, 2015, e Sartoretto et al, 2015, a espessura da amostra deve ser de aproximadamente 20 a 30µm, sem nenhum tipo de sobreposição da imagem (roscas e osso) quando visto em microscópio.…”

“…Segundo Soares et al, 2015, e Sartoretto et al, 2015, a espessura da amostra deve ser de aproximadamente 20 a 30µm, sem nenhum tipo de sobreposição da imagem (roscas e osso) quando visto em microscópio. Espessura a qual é dada através da politriz com uso das lixas de várias granulações (SARTORETTO et al, 2015, SOARES et al, 2015CALVO-GUIRADO et al, 2013;SOBOL et al, 2011;OLIVEIRA et al, 2009;STENPORT et al, 2008).…”