Figure 6: HRP-GST-ABD can effectively and efficiently amplify the signal in TSA-based immunostaining.

(A) Anti-acetylated tubulin primary antibodies (mouse) are used to detect nerve fibers in sectioned Xenopus samples. The forebrain region is sectioned transversely and the nerve fibers are visualized with HRP-GST-ABD and tyramide-Alexa 488 (green). The nuclei are stained with DAPI in blue. The nerve fibers in the forebrain are clearly stained in green (A’) and the ciliary axonemes are specifically stained only in multiciliated cells (A”). The scale bars in a’ and a” are 20 μm and 5 μm, respectively. (B) Anti-phospo-histone H3 primary antibodies (rabbit) are used to detect proliferating cells in green. The tubulins are stained in red with anti-tubulin antibodies (rabbit) and the Alexa-555 conjugated anti-rabbit secondary antibodies as a counterstain after the TSA reaction. Note that the Alexa-555 conjugated anti-rabbit secondary antibodies also detected the anti-phospho-histone H3 primary anti-bodies, which are used for the TSA reaction, and the green signals amplified by HRP-GST-ABD precisely overlapped with the red signals only in the nuclei (B’,B”). The scale bars in B’ and B” are 20 μm and 5 μm, respectively. (C) An anti-fibronectin primary antibody (mouse) was used to detect the extracellular matrix. The scale bars in (C’,C”) are 20 μm and 5 μm, respectively. (D,E) The anti-phospho-histone H3 primary antibody (rabbit, D) or anti-acetylated tubulin primary antibody (mouse, e) are used again to stain whole mount embryos. An anti-actin antibody is used to visualize the cell boundaries in red (D). (D’,D”) show the confocal images in indicated sectioning planes. (E) HRP-GST-ABD successfully penetrates through whole mount embryo body and specifically amplified the signals of target molecules in whole mount samples. Acetylated-tubulin antibodies also detected multiciliated cells in the epidermis in addition to the nerve fibers. The scale bars in d and e are 20 μm. All control images obtained by using the regular HRP-conjugated secondary antibodies (rabbit and mouse) are presented in the supporting Figures (S10–12).