Journal: Nature biotechnology

Article Title: Simultaneous protection of tissue physicochemical properties using polyfunctional crosslinkers

doi: 10.1038/nbt.4281

Figure Lengend Snippet: ( a-c ) Representative FP signals of brain slices subjected to thermal treatment after processed with fixatives (PFA, GA, or P3PE). Brain slices expressing EGFP (Thy1::EGFP M-line), YFP (Thy1::YFP H-line), or tdTomato (PV-Cre / loxP-tdTomato) were used. Scale bar = 1 mm (a), 100 μm (b,c) ( d ) FP signal retention after the same heat treatment in brain sections preserved with difference fixatives and CLARITY. N=3 tissues. ( e ) GFP signal retention from M-line slices after exposure to organic solvents and detergents. MeOH, methanol; THF, tetrahydrofuran; TBA, tert -butyl alcohol; BABB, one part benzyl alcohol and two parts benzyl benzoate; TX100, Triton-X100; BDEA, butyldiethanolamine. N=3 tissues. ( f ) Fluorescence images of neurons virally labeled with RV-hSyn-mOrange-p2A-PSD95-GFP in GA and SHIELD tissue. Scale bars = 100, 10, and 1 μm ( left to right ). ( g ) Fluorescence intensity profiles of PSD95-GFP (green) and mOrange (red) signals along the dotted lines in f . ( h ) Tissue autofluorescence from various excitation wavelengths. N=3 tissues. ( i ) Representative images comparing the immunofluorescence of key cell-type antibodies in tissues prepared by various tissue processing methods. Scale bar = 20 μm. FoxP2, forkhead box protein P2; CR, calretinin; PV, parvalbumin; NeuN, neuronal nuclei; GFAP, glial fibrillary acidic protein; Iba1, ionized calcium-binding adapter molecule 1. The same imaging and display settings were used for each antibody. ( j ) Signal to noise ratios (SNR) of immunofluorescence in i normalized to the SNR of PFA control. N=3 tissues. ( k, l ) SHIELD preserves endogenous YFP fluorescence during multiple rounds of immunostaining and destaining. ( k ) Overlay of multi-round immunostained images. Scale bar = 100 μm. ( l ) Images from individual rounds. Scale bar = 100 μm. ( m ) Representative heatmaps of fluorescence in situ hybridization (FISH) of total mRNAs by (dT) 50 -Cy3 in cleared PFA, EDC-CLARITY, GA, and SHIELD tissues. Scale bar = 100 μm. ( n ) Fluorescence intensities of dT 50 -Cy3 FISH normalized to the signal of uncleared PFA tissues (Control), N=3 tissues. ( o ) FISH HCR against three mRNAs in SHIELD tissue. Scale bar = 1 mm ( left ) or 50 μm ( right panels ). ( p ) Dual labeling of c-Fos protein and mRNA in SHIELD tissue from a mouse foot shocked (FS) twice at 35 and 5 minutes before sacrifice. Scale bar = 10 μm. ( q ) Uniform preservation of transcripts in a SHIELD-processed brain hemisphere cleared with stochastic electrotransport (SE). See . Scale bar = 2 mm ( left ) or 100 μm ( right panels ). Mean +/− standard error mean was used to plot all the bar graphs. One-way ANOVA, Turkey’s multiple comparison test, * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: SHIELD-fixed organs were cleared passively for a couple of weeks (14 days at 45°C for mouse brain hemisphere) in SDS-based clearing buffer (300 mM SDS, 10mM sodium borate, 100mM sodium sulfite, pH 9.0), or rapidly cleared (<3 days for mouse brain hemisphere) using stochastic electrotransport (SmartClear Pro, LifeCanvas Technologies).

Techniques: Expressing, Fluorescence, Labeling, Immunofluorescence, Binding Assay, Imaging, Control, Immunostaining, In Situ Hybridization, Preserving, Comparison

Journal: Nature biotechnology

Article Title: Simultaneous protection of tissue physicochemical properties using polyfunctional crosslinkers

doi: 10.1038/nbt.4281

Figure Lengend Snippet: A series of xy-plane images showing uniform (dT) 50 -Cy3 FISH signal in a SHIELD-processed block. A 1 mm-thick block is dissected from an intact mouse hemisphere preserved by SWITCH-mediated SHIELD followed by active delipidation using stochastic electrotransport. Z-step size: 10 μm. This result was confirmed in 3 independent trials with similar results.

Article Snippet: SHIELD-fixed organs were cleared passively for a couple of weeks (14 days at 45°C for mouse brain hemisphere) in SDS-based clearing buffer (300 mM SDS, 10mM sodium borate, 100mM sodium sulfite, pH 9.0), or rapidly cleared (<3 days for mouse brain hemisphere) using stochastic electrotransport (SmartClear Pro, LifeCanvas Technologies).

Techniques: Blocking Assay