Fig. 3: Development and applications of deep immunostaining using thermostabilized antibody–Fab complex.
From: Antibody stabilization for thermally accelerated deep immunostaining
a, Tolerance of SPEARs to the duration and condition of ThICK staining at 55 °C. Upper panels: x–z view of mouse spinal cords that have been ThICK-stained with ChAT SPEARs. Scale bars, 50 μm. Lower panels, example cells from different depths. Scale bars, 10 μm. Color bar, pixel intensity. b, From left to right: homogeneity of pixel intensity mean, variability (that is, s.d.), and signal to noise ratio (SNR) by depth for various durations of ThICK staining (in hours). c, ThICK staining of formaldehyde-fixed (left panel, 55 °C for 1 h) and SHIELD-protected (right panel, 55 °C for 16 h) tissues with endogenous fluorescence. Scale bars, 50 μm. d, Antibodies compatible with SPEAR synthesis and ThICK staining. Green, Alexa Fluor 488; red, Alexa Fluor 594; cyan, Alexa Fluor 647. MIP, maximum intensity projection; PV, parvalbumin; TH, tyrosine hydroxylase; VIP, vasoactive intestinal peptide. Scale bars, 20 μm. e, ThICK staining with NanoSPEARs. Scale bars, 20 μm. f, Optimization of ThICK-staining buffer composition with respect to SPEARs intravascular precipitates per unit imaged tissue volume. The experiment was repeated two more times for control (0.3% Triton X-100) and 1 M GnCl. The error bars represent s.d. P = 0.0216, two-sided unpaired t-test with Welch’s correction. g, Pyridine (py)-catalyzed P3PE-crosslinking reaction. h, A higher concentration of py is associated with more conversion of precursor to product. i, A total of 61.8 mM py accelerates crosslinking when compared with non-catalyzed control by SDS–PAGE. Inset: results of 1–8 h reaction time. ***P ≤ 0.01 at 4 and 8 h reaction (multiple two-sided t-test with multiple comparisons adjustment), n = 3 replicates; data are given as mean ± s.d. j, Functional assay based on hot-start PCR for testing py-catalyzed synthesized SPEARs (SPEARpy) and agarose gel analysis of the PCR product in the lower panel. k, Functional activity of Taq SPEAR versus Taq SPEARpy in inhibiting PCR product formation, comparing SPEARs used directly after synthesis with those pre-heated at 55 °C for 16 h. The experiment was repeated six times and data are given as mean ± s.d. n.s., not significant. ***P ≤ 0.001 (Tukey’s multiple comparison test, two-sided). l, Comparison of staining qualities of ChAT SPEAR and ChAT SPEARpy. Left panels, representative images of staining with ChAT SPEARs. Scale bars, 20 μm. Right panel, signal to background ratio along the axes of representative cells (white rectangles). Data are given as mean (solid lines) ± s.d. (shaded regions). Lighter lines represent normalized intensity profiles of individual cells. m, Optimized ThICK staining with ChAT SPEARpy (red) in a SHIELD-protected sample with endogenous neuronal GCaMP6f (green). Precipitates were identified and highlighted in white. Scale bars, 50 μm.
