Figure 3. Comparison of antibody-based and DNA-based amplification.
(A) Workflow for TSA reaction. DIG-labeled probes are hybridized to target mRNA. HRP conjugated anti-DIG antibodies bind to hybridized probes and are detected by TSA.
(B) Workflow for HCR reaction. Initiator-labeled probes are hybridized to target mRNA. In a second step, initiator sequences hybridize to toehold of fluorophore bearing hairpins, starting a chain reaction of hairpin assembly.
(C-D) In situ hybridization for somatostatin mRNA in CLARITY tissue. Above, z = 100 µm, 200 µm, and 300 µm into CLARITY tissue volume of mouse cortex using traditional ISH (C) or hybridization chain reaction (D). Scale bars, 100 µm. Below, yz-subsections of CLARITY volume and 3D rendering of 1mm sections. Arrowheads indicate the z-location of the sections above. Arrows indicate the tissue surface. Due to high surface background, the top 130 µm of tissue are not shown for the 3D rendering in (C). Scale bars, 100 µm (left), 300 µm (right).
(C) DIG-labeled oligonucleotide probes detected with anti-DIG antibody (2 days) and TSA. Somatostatin expressing cells can be detected, but surface has high background and signal diminishes deeper in the tissue.
(D) Initiator-labeled oligonucleotide is detected with HCR reaction (1 day), resulting in more uniform staining.
(E) Number of cells as a function of tissue depth after local thresholding and cell segmentation on each imaging plane, 10 µm z-interval. High surface background in TSA reaction yields a large number of putative false positives 0-75 µm into the tissue section. Detection with HCR amplification shows a more uniform labeling of cells, comparable to the distribution of somatostatin cells in a genetically encoded reporter mouse (Sst-TFP).
(F) Ratio of signal to background as a function of depth in tissue, calculated from ratio of mean signal intensities segmented in (F) to the mean background intensity. For (E-F), No Probe, n=3; Scrambled Control, n=3; TSA, n=5; HCR, n=9; Sst-TFP, n=4. P<0.05, Kruskal-Wallis test on mean ratio over entire depth. All error bars indicate SEM. See also Figure S2