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. 2020 Feb 14;12:6. doi: 10.3389/fnsyn.2020.00006

Figure 6.

Figure 6

Example results using recommended dilutions of two-helper combination. (A–H) Use of the two-AAV combination at 1:200 and 1:20 dilutions (see main text) labeled many presynaptic neurons. (A) Injection site in S1. Green = anti-EGFP staining, blue = mTagBFP2, red = mCherry. Individual channels from this field are shown in panels (E–H). (B) Many labeled neurons were found in ipsilateral S2. (C) Many labeled neurons were found in ipsilateral thalamus (VPL, VPM, and Po). (D) Many labeled neurons were found in contralateral S1. (E–H) Individual channels from the field shown in panel (A). (E) Anti-parvalbumin staining (not shown in panel A). (F) Anti-EGFP staining, indicating expression from the first, Cre-dependent AAV. Note that, at this dilution, the EGFP signal is quite dim even with immune amplification. (G) mTagBFP2, indicating expression from the second, tTA-dependent AAV. (H) mCherry, indicating the presence of the ΔG RV. (I–L) Injection site after using two-helper combination at 1:200 and 1:20 (see main text): few mCherry-labeled cells are seen. (I) Overlay of (J–L). (J) Anti-EGFP staining: no signal is visible, even with amplification. (K) mTagBFP2 signal. A few blue cells are seen even at these dilutions. (L) mCherry expressed by RV. Scale bar in (A): 200 μm, applies to all panels.