Fig. 2.

Ultrafast and homogeneous tag-based tissue staining. (A) Direct comparison of immunostaining and chemical-labeling protocols. In these ethograms the length of individual steps is proportional to the time required, and each black square represents a manual interaction. Chemical labeling is >100× faster (∼1 h vs. >100 h) and requires half as many (8 vs. 15) manual handling steps of the sample. (B) Staining time course of GH146 projection neurons using immunostaining against membrane-targeted GFP (Top Row), chemical labeling using SNAP-tag (Middle Row), or Halo-tag (Bottom Row). The z-maximum intensity projections from 3D confocal stacks are shown. Incubation times are indicated for primary antibody or chemical substrates (GFP antibody, SNAP BG-549, Halo-TMR, nc82-antibody). Note that incubation with secondary antibodies was for 2 d. (C) Staining time course of the nc82/Brp neuropil marker using immunostaining against Brp protein (Upper Row) or chemical labeling using SNAP-tag (Lower Row). Single coronal confocal slices through the center of the brain are shown. Note that incubation with secondary antibodies was for 2 d. Also note that background labeling increases with longer substrate incubation times (asterisk). (Scale bars in B and C: 50 µm.) (D) Quantification of signal intensity over time in GH146-Gal4 brains labeled with antibody vs. chemical labeling. Secondary antibodies for GFP immunostaining were incubated for 2 d (myrGFP-2day) or for the same duration as primary antibodies (myrGFP-equal). Fluorescence was quantified using a GH146 mask (n = 5–8 brains per condition) (Fig. S3B and SI Materials and Methods). (E) Quantification of labeling intensity (SI Materials and Methods) at different depths from the brain surface in brains labeled with nc82 antibody or chemically labeled (using Brp-SNAP). Secondary antibodies for nc82 immunostaining were incubated for 2 d (nc82-2day) or for the same duration as primary antibodies (nc82-equal). Five different time points are shown.