Whole-mount immunostaining is a powerful tool to study protein distribution in embryos and other bulky samples (e.g. Klymkowsky and Hanken,1991; Linask and Tsuda,2000; Müller,2008). In Xenopus embryos, tissue formation could be monitored by use of differentiation markers such 12/101 (somite and skeletal muscle, Kintner and Brockes,1985) or 3G8 and 4A6 (pronephros, Vize et al.,1995), the activity of cell signaling by antibodies against components of different signaling pathways (e.g. Christen and Slack,1999; Schneider et al.,1996), and ectopically expressed proteins by antibodies against protein tags such as GFP, myc-tag, etc. Due to the opacity of early Xenopus embryos, however, the resolution of such whole-mount preparations is rather limited. To gain more detailed insight into tissue organization in relation to specific protein expression, the analysis of histological sections is required. This could be achieved by the embedding of immunolabeled embryos into Technovit 7100 for sectioning. This method combines, for example, whole-mount immunofluorescence with resin embedding and serial sectioning (Table 3). The method works best with samples that are fixed with Dents fixative (Dent et al.,1989). Although the fixation is rather harsh and results in strong cytoplasmic extraction (Kurth,2003; see also Fig. 1c,d), this morphological defect is less evident in fluorescent images (Figs. 8 and 9). Whole-mount immunofluorescence in combination with resin embedding has been mainly used for the visualization of cell adhesion molecules such as cadherins (Angres et al.,1991; Kurth et al.,1999; Müller et al.,1992,1993; Münchberg et al.,1997; Ogata et al.,2007a,b), catenins (Kurth et al.,1996,1999; Schneider et al.,1993), integrins (Gawantka et al.,1992,1994; Joos et al.,1995,1998; Müller et al.,1993), and tight junctional proteins (Fesenko et al.,2000), for cytoskeletal proteins (Kurth et al.,2000) and for different nuclear antigens (e.g. David et al.,1998; Ellinger-Ziegelbauer and Dreyer,1993; Schwab and Dreyer,1997). The procedure is not applicable to all antigens, because proteins that are not firmly attached to cellular structures such as cytoskeleton, membranes, or the nucleus often get extracted during Dent's fixation. Fixation in 4% PFA before transfer into Dent's fixative improves the preservation of such proteins, but in that case antibody incubation, washing, dehydration, and infiltration steps have to be increased, which results in a preparation protocol lasting nearly 2 weeks (Kurth,2003,2005).
Figure 8. Whole-mount Immunofluorescence followed by embedding in Technovit 7100 and analysis of 2 to 5 μm thick sections. (a) Dorsoanterior mesoderm (mes) migrating along the blastocoel roof (bcr); ect, ectoderm; end, endoderm. Antibodies: anti-fibronectin (FN, red), XB/U-cadherin (green). Nuclei counterstained with DAPI. (b) Xenopus animal cap (explanted at stage 9, fixed at stage 22), overview. (c) same cap at higher magnification. Antibodies: anti-β-catenin (P14L, red), anti-phospho-histone H3 (red, due to the overlap with DAPI it appears magenta), anti-tubulin (green). Nuclei counterstained with DAPI. (d) Fluorescence of membrane-anchored GFP (memGFP) in a tailbud embryo after fixation, embedding, and sectioning. memGFP-mRNA was injected into one blastomere at the two-cell stage; epi, epidermis; myo, myotome; not, notochord; nt, neural tube. (e) Section through the dorsal side of stage 11 gastrula. At the 16-cell stage, memGFP-mRNA and rhodamin-dextran were injected into dorsoanterior animal and vegetal cells, respectively, and the embryos cultured until stage 11. After fixation, they were embedded in Technovit 7100, sectioned and scored for GFP (green) and rhodamine (red). (f) Section through the smooth muscle layers of the Xenopus adult intestine. Smooth muscle cells are surrounded by thin layer of ECM and strongly express β1-integrin (green); bv, blood vessel; mu, smooth muscle; sub, submucosa. (g–i) Axolotl embryos (stage 24) stained with anti-actin (green) and anti-β-Catenin (red). (g) Somite (som), epi, epdermis. (h) Notochord (not), neural tube (nt) and archenteron (arch). (i) Large endodermal cells.
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Whole-mount immunofluorescence in combination with Technovit embedding and semithin sectioning (1–2 μm) reveals a convenient balance of resolution and signal intensity (Figs. 8 and 9). It is also very useful to monitor in vivo markers such as GFP or fluorescent dextrans after embedding and sectioning of fixed samples. Mem-GFP, for example, retains its fluorescence even after fixation, dehydration, and plastic embedding (Fig. 8d,e). This way, different sets of in vivo markers can be combined with antibody stainings. Finally, the immunofluorescence in one section can be correlated to the overall organization of the sample revealed by histochemical staining with TB/Borax of the adjacent section. Since the sections are just 1 to 2 μm apart from each other, this correlative TB/Borax and fluorescence imaging makes it possible to relate tissue organization with protein distribution in the very same area of the embryo (Fig. 9).