Immunofluorescence and RNA in situ hybridization are microscopy techniques that are extensively used for the localization of antigens and mRNAs with single cell resolution. Both techniques include a preparatory fixation and permeabilization steps. Fixation is a procedure intended to stabilize the cell structure, preserving it as closely as possible to that of the living cell. It may be achieved by precipitating or additive fixatives. Precipitating fixatives, such as cold organic solvents (methanol, acetone, ethanol, or mixtures of them), denature proteins and hence disrupt their three-dimensional (3D) structure. In addition, they provoke dehydration and cell shrinkage. Under these conditions, cell structure is poorly preserved. However, antigenic sites and mRNAs are not covalently modified and, thus, may be available to bind antibodies and mRNA probes, respectively. Additive fixatives, such as formaldehyde and glutaraldehyde, cross-link proteins preserving their native 3D structure (Hayat, 2002). Formaldehyde is a gas that dissolves in water to 37–40%, and reacts with it to form methanediol (Kiernan, 2000). As polymerization of this compound increases, its fixation properties decrease. For this reason, monomeric formaldehyde obtained from paraformaldehyde is commonly used to prepare cells for immunocytochemical procedures. Aldehyde-driven cross-linking is achieved by formation of methylene bridges between amino acids (Puchtler and Meloan, 1985; Kiernan, 2000; Sutherland et al., 2008) or between amino acids and nucleic acids (Dedon et al., 1991; Orlando et al., 1997; Spencer and Davie, 2002; Schmiedeberg et al., 2009). Formation of methylene bridges by formaldehyde appears to occur in two steps. The first step involves the formation of highly reactive methylol groups by the reaction of methylene hydrate with: (a) reactive groups, mainly NH2 groups of N-terminal amino acid residues and the side chains of arginine, cysteine, histidine, and lysine, and (b) cytosine amino groups of nucleic acids. During the second step, the reaction with an another amino group leads to a condensation reaction with the formation of methylene bridges between adjacent polypeptide chains. Additive fixatives covalently modify proteins and mRNAs that become less able to bind antibodies and mRNA probes. To solve this problem, cell and tissues must be permeabilized before, during or after fixation. Permeabilization is a process by which membranes are solubilized by detergents and/or enzymes allowing antibodies and RNA probes to reach the antigenic sites and native mRNAs, respectively (Puchtler and Meloan, 1985; Orlando et al., 1997; Kiernan, 2000; Hayat, 2002; Spencer and Davie, 2002; Renshaw, 2007). In the case of immunofluorescence, permeabilization and fixation are followed by incubation in the primary antibody and then its recognition by a secondary antibody linked to a fluorophore. For mRNA in situ hybridization, fixed and permeabilized samples must first be hybridized with a labeled RNA probe after which the hybridization product is visualized by immunofluorescence or immunocytochemistry. This last process is called staining.
Preparation of cells or tissues for immunofluorescence and mRNA in situ hybridization thus involve three main steps: fixation, permeabilization and staining. Because the first two steps may be performed in a different sequence, separately or together, one may consider three modalities for applying these techniques: (a) Permeabilization, fixation, and staining. This is the less common modality but one that has successfully been used for the immunofluorescence staining of the cytoskeleton of leech embryos (Fernández et al., 1990, 1994; Fernández and Olea, 1995). In this case, permeabilization is performed first using an extraction buffer including a nonionic detergent, protein stabilizers and antiproteases (Fernández et al., 1998; Cantillana et al., 2000). Fixation is performed afterward using paraformaldehyde-Triton-X-100. This detergent is also added to the antibody staining and washing solutions. (b) Fixation, permeabilization, and staining. This is the most common procedure for immunofluorescence and mRNA in situ hybridization in which samples are first fixed with paraformaldehyde (Schulte-Merker et al., 1992; Strähle and Jesuthasan, 1993; Strähle et al., 1993; Jesuthasan, 1998; Thisse and Thisse, 2008) or formaldehyde (Henrique et al., 1995; Streit and Stern, 2001; Kang et al., 2002). Small amounts of detergent, glutaraldehyde, calcium chelators, Mg salts or cytoskeleton stabilizers may be added to either of the fixatives (Hammati-Brivanlou and Harland, 1989; Harland, 1991; Stachel et al., 1993; Henrique et al., 1995; Leung et al., 2000; Streit and Stern, 2001; Dekens et al., 2003). In the majority of cases, permeabilization is achieved by treating the fixed embryos with detergents, cold organic solvents or a mixture of both (Hammati-Brivanlou and Harland, 1989; Harland, 1991; Schulte-Merker et al., 1992; Stachel et al., 1993; Leung et al., 2000; Thisse and Thisse, 2008). For mRNA in situ hybridization, proteinase K or pronase E are used for further permeabilization (Allende et al., 1996; Kang et al., 2002). After permeabilization, cells or tissues are subjected to immunostaining or mRNA in situ hybridization. Antigen retrieval after fixation may be also achieved by heat (Shi et al., 2001; Hayat, 2002; Renshaw, 2007) and this procedure is compatible with mRNA in situ hybridization and detection of fluorescent proteins in transgenic fish lines (Inoue and Wittbrodt, 2011). (c) Fixation/permeabilization and staining. In this case, fixation/permeabilization is carried out simultaneously and can be achieved by using cold organic solvents such as methanol, that fix proteins while keeping the antigenic sites of proteins and the nucleic acids available to bind antibodies and hybridization probes, respectively. To further improve accessibility to antigenic sites, detergents, calcium chelators, or proteolytic enzymes may be also added to the fixative or during the washing and staining of the samples (Wühr et al., 2008). In other cases, a detergent is added to the aldehyde fixative that was prepared with stabilizers or in an assembly buffer (Gard, 1991; Schroeder and Gard, 1992; Pelegri et al., 1999).
In this study, we show that combined fixation/permeabilization, using a mixture of formaldehyde or paraformaldehyde with a short C-chain aliphatic carboxylic acid (particularly glacial acetic acid), that acts as a permeabilizer, allows improved preparation of embryonic cells and tissues for immunofluorescence and mRNA in situ hybridization. Detergents, organic solvents, proteinases, and refixation steps were omitted. With this tool, we developed protocols for immunostaining and mRNA in situ hybridization of different vertebrate and invertebrate embryos. The advantage of using these protocols is discussed.