Ultrafast tissue staining with chemical tags

Abstract

Genetically encoded fluorescent proteins and immunostaining are widely used to detect cellular and subcellular structures in fixed biological samples. However, for thick or whole-mount tissue, each approach suffers from limitations, including limited spectral flexibility and lower signal or slow speed, poor penetration, and high background labeling, respectively. We have overcome these limitations by using transgenically expressed chemical tags for rapid, even, high-signal and low-background labeling of thick biological tissues. We first construct a platform of widely applicable transgenic Drosophila reporter lines, demonstrating that chemical labeling can accelerate staining of whole-mount fly brains by a factor of 100. Using viral vectors to deliver chemical tags into the mouse brain, we then demonstrate that this labeling strategy works well in mice. Thus this tag-based approach drastically improves the speed and specificity of labeling genetically marked cells in intact and/or thick biological samples.

Keywords: fluorescence microscopy; immunohistochemistry; neural circuits; protein labeling.

Fig. 1.

Expression of chemical tags in the fly brain. (A) Chemical-labeling chemistries used in this study (adapted from refs. –, ; see text for description). (B) Expression patterns of membrane-targeted tags in the Drosophila central brain. The panel is arranged in a three-row by four-column grid. Rows represent Gal4 driver lines; columns represent reporter constructs. In the far-left column, nc82 neuropil counterstaining is shown in magenta. The fluorescent substrates used are indicated for each tag. Note that although myrGFP, myrSNAP, and myrHalo are targeted attP insertions, CD4-CLIP is a P element insertion. Because of positional effects, the CD4-CLIP reporter shown here labels the bilateral anterior paired lateral neuron when crossed to GH146-Gal4 (asterisk). (C) Brains of GH146-Gal4 animals expressing either myrSNAP or myrGFP stained with GFP antibody or with fluorescent BG-488, BG-549, or BG-647 substrates, as indicated. (D) Comparison between native GFP signal and chemical labeling in brains of Mz19-Gal4 > myrGFP, myrSNAP (Upper) and Mz19-Gal4 > myrGFP, myrHalo (Lower) animals. (Scale bars: 50 µm.)

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.

Fig. 3.

Key applications for chemical labeling in Drosophila neurobiology. (A) Comparison between epitope-tagged synaptic markers (HA-tag, mCherry) and synaptically targeted chemical tags (SNAP-tag, CLIP-tag). Confocal slices from the brain of a fru^Gal4> SytHA, SytCLIP (presynaptic markers) animal (Left) and a fru^Gal4> TLN-mCherry (DenMark), TLN-SNAP (somatodendritic markers) animal (Right) are shown. (Scale bar: 50 µm.) (B) Simultaneous labeling of presynaptic sites and the somatodendritic compartment of DA1 projections neurons using Mz19^Gal4. (Scale bar: 50 µm.) (C) Map of the brp-SNAP knockin. (D) nc82 and Brp-SNAP signals colocalize in a brp-SNAP/⁺ brain simultaneously labeled with BG-549 and nc82 immunostaining. Single coronal slices through the middle of the brain (Left) and through a deconvolved image stack of the DA1 glomerulus (Right) are shown. Note the more even Brp-SNAP staining in the center of the brain. (Scale bars: 50 µm, Left; 2 µm, Right.) (E) Deconvolved, confocal z-stack images from the MB calyx region of a brp-SNAP, Mz19-Gal4 > CLIP-syb, myrHalo brain triple-labeled with fluorescent BG-488, BC-547, and Halo-SiR substrates. Colocalization analysis of SNAP-tag– and Halo-tag–labeled regions reveals presynaptic sites (Brp puncta) within PN terminals (see also Movie S1). (Scale bar: 10 µm.) Montages show three individual, triple-labeled projection neuron terminals (Right, 1–3) derived from the indicated boxed regions. One selected optical section (marked with an asterisk) is also shown with Brp-SNAP only (green), revealing that puncta are localized to the surface of each terminal. (F) Chemically labeled neurons can be registered successfully onto a template using the Brp-SNAP neuropil counterstaining, thus allowing direct comparison with imaged neurons from other sources, such as those derived from stochastic labeling (Flp-out), PA-GFP tracing, whole-cell recordings, or sparse driver lines, (e.g., Mz19-Gal4). (Scale bar: 50 µm.) (G) Overlay of DA1 projection neurons (green) from a chemically labeled brp-SNAP, Mz19-Gal4 > myrHalo brain (Left) overlaid with a dye-filled third-order olfactory neuron (cyan) from an nc82-stained brain (Right) after registration. Note the overlap between axon terminals and dendritic arbor (white arrowheads).

Fig. 4.

Chemical labeling of the mouse brain. (A) Virus construct for chemical labeling of mouse brain. (B) A 200-µm coronal vibratome slice from a mouse brain injected with AAV-myrSNAP into hippocampus, stained with BG-549 (SI Materials and Methods). Insets show counterstaining against the neuronal marker NeuN (Left) and individual labeled neurons (Middle, arrowheads). (Scale bars: 250 µm in overview panels; 100 µm in Insets.) (C) Overview scan of a coronal vibratome slice from mouse brain unilaterally injected with AAV-myrSNAP into hippocampus and stained with BG-549 (Left). Numbered boxed areas 1–3 are expanded on the right. Single contralateral SNAP-positive projections are visible on the noninjected side (C, 2, Lower Right). (Scale bar: 500 µm.) (D) Background labeling from BG-549 is very low in zones devoid of transfected neurites (area below the dashed line; see box in B, Right). (Scale bar: 100 µm.) (E) Volume rendering of the area indicated by the dashed rectangle in C, 2, Lower Right). The high signal-to-noise ratio of BG-549–labeled processes allows facile 3D reconstruction. Four separate traced neurites are shown in different colors. (Scale bar: 200 µm.) (F) Side views of maximum-projected 3D confocal stacks of brain slices labeled with myrSNAP (Left, samples from B) or stained with anti-Tau antibody (Right) are shown with incubation times indicated. Two individual neurites are highlighted in magenta. (G) The intensity profile along the z axis for individual SNAP-labeled fibers was quantified (n = 9–11 per condition) (SI Materials and Methods). Then the normalized intensity profile was plotted for each labeling time (Left). For Tau staining, image intensity was quantified directly, because all neurons are labeled (Right) (SI Materials and Methods).

Fig. 5.

Chemical labeling of genetically defined neurons in mouse brain. (A) Virus construct for conditional (Cre-dependent) chemical labeling of mouse brain. (B) Coronal 200-µm vibratome slice from PV-Cre mouse brain injected with AAV-myrSNAP-CON, stained with BG-549. (C and D) Single neurons labeled with BG-549 (Scale bar: 250 µm) (C) and stained with anti-PV antibody (Scale bar: 50 µm) (D). (E) No-substrate control for brain injected with AAV-myrSNAP-CON (coronal vibratome slice through the hippocampus). (Scale bar: 50 µm.)