Journal: Scientific Reports

Article Title: ADAPT-3D:accelerated deep adaptable processing of tissue for 3-dimensional fluorescence tissue imaging for research and clinical settings

doi: 10.1038/s41598-025-16766-z

Figure Lengend Snippet: ADAPT-3D maintains tissue integrity and fluorescence intensity for accurate 3D imaging without excessive lipid removal. ( A ) Stereoscope images of 1-mm fixed brain slice from a LysM Cre ;TdTomato fl/fl mouse before treatment (top), immediately after 12 h in CUBIC-L at 37 °C (middle), and after washing out of CUBIC-L buffer (bottom). ( B ) Matched brain slice from the contralateral hemisphere to that in A before treatment (top), captured immediately after 12 h in ADAPT:PDL (middle), and after washing out of ADAPT:PDL (bottom). ( C ) Stereoscope image of CUBIC processed 1 mm section from A after immersion in CUBIC-R+(M) for 4 h (left) and the ADAPT-3D processed 1 mm section from B after immersion ADAPT: RI for 4 h (right). ( D ) Tile scanned confocal image of the first in focus plane of the CUBIC processed section (left) and of the ADAPT-3D processed section (right) acquired with matched acquisition settings and displayed with equivalent scaling. ( E ) The area of CUBIC and ADAPT-3D processed sections measured from tiled confocal fluorescent images using imageJ software. ( F ) The mean fluorescence intensity of anti-Histone-ATTO488 and ( G ) of endogenous TdTomato in tiled confocal images acquired from sections processed with either CUBIC or ADAPT-3D (two-tailed unpaired t-test, standard error of mean bars, *p-value ≤ 0.05, *** p-value ≤ 0.001, ****p-value ≤ 0.0001). ( H ) Zoomed in view of the lateral and 3rd ventricle in the CUBIC processed section from D that was increased in brightness to be visible and ( I ) of the choroid plexus in the lateral ventricle of the ADAPT-3D processed section from D. ( J ) The leptomeninges on the cortical edge of a section processed with CUBIC vs. ( K ) a section processed with ADAPT-3D. * in panel J indicates 1 of multiple sites of leptomeningeal tearing. ( L ) Stereoscope image of white matter in matched sections where one was treated for 3 h with CUBIC-L at 37 °C (top) and the other with ADAPT:PDL for 3 h (bottom) that were washed out of their respective delipidation buffers. ( M ) Fixed peritoneal fluid cells from LysM cre ;Abca1 fl/fl Abcg1 fl/fl mice untreated (left) or incubated in ADAPT:PDL for 15 min (right) and stained with LipidSpot 610. ( N ) Representative image of CellBrite Orange staining for membrane lipids in fixed cells from peritoneal fluid without delipidation (left) or after a 15-minute incubation in ADAPT:PDL (right). ( O ) Representative image of EEA1 staining for endosomes in fixed cells from peritoneal fluid without delipidation (left) or after a 15-minute incubation in ADAPT:PDL (right) where combined endosomal and nuclear staining is shown as an inset.

Article Snippet: 3D visualization was visualized on Imaris software (Bitplane Inc.) on v10.1.1 software.

Techniques: Fluorescence, Imaging, Slice Preparation, Software, Two Tailed Test, Incubation, Staining, Membrane

Journal: EMBO Molecular Medicine

Article Title: AAV gene therapy rescues hearing and balance in a model of CLIC5 deafness

doi: 10.1038/s44321-025-00275-7

Figure Lengend Snippet: ( A ) 3D surface projection of IHCs and OHCs from the cochlear 32 kHz region of Clic5 +/− , Clic5 −/− and Clic5 −/− injected with scAAV. Clic5 at 12 weeks, stained with a CLIC5 (red) and actin (gray). ( B ) Representative confocal images of IHCs from the apical turn of Clic5 +/− and Clic5 −/− injected with scAAV. Clic5 at 12 weeks, stained with a CLIC5 (red), actin (gray), and FLAG tag (green). ( C ) Representative confocal images of IHCs and OHCs from the cochlear 8 kHz region of Clic5 +/− , Clic5 −/− , and Clic5 −/− injected with scAAV. Clic5 at 12 weeks, stained with actin. ( D ) Stereocilia length measurements of IHC at different regions of the cochlea, n = 3 for Clic5 +/− , Clic5 −/− , and Clic5 −/− injected with scAAV. Clic5 . ( E ) Quantification of OHC survival at different regions of the cochlea, n = 3 for Clic5 +/− , Clic5 −/− , and Clic5 −/− injected with scAAV. Clic5 . ( F ) Quantification of IHC survival at different regions of the cochlea, n = 3 for Clic5 +/− , Clic5 −/− , and Clic5 −/− injected with scAAV. Clic5 . ( G ) High-magnification scanning electron microscopy images of outer and inner hair bundles from the cochlea middle-turn of the Clic5 −/− and Clic5 −/− injected with scAAV. Clic5 at 12 weeks. ( H ) Representative images of IHCs and OHCs at 12 weeks at different regions of the cochlea of Clic5 −/ − injected with scAAV. Clic5 . ( I ) ABR thresholds at 4 weeks of Clic5 +/− ( n = 11), Clic5 −/− ( n = 10), and Clic5 −/− injected with scAAV. Clic5 ( n = 10). ( J ) ABR thresholds at 8 weeks of Clic5 +/− ( n = 11), Clic5 −/− ( n = 11), and Clic5 −/− injected with scAVV. Clic5 (n = 7). ( K ) ABR thresholds at 12 weeks of Clic5 +/− ( n = 10), Clic5 −/− ( n = 10), and Clic5 −/− injected with scAAV. Clic5 ( n = 7). ( L ) DPOAE thresholds at 4 weeks of Clic5 +/− ( n = 12), Clic5 −/ − ( n = 12), and Clic5 −/− injected with scAAV. Clic5 ( n = 10). ( M ) ABR thresholds at 3-time points of Clic5 −/− injected with scAAV. Clic5: 4 weeks ( n = 10), 8 weeks ( n = 7), and 12 weeks ( n = 7). ( N ) Freezing behavior duration in a fear conditioning assay at 12 weeks of Clic5 +/− ( n = 14), Clic5 − /− ( n = 14), and Clic5 −/− injected with scAAV. Clic5 ( n = 6). ( O ) Expression rates of scAAV. Clic5 across the cochlea as a function of viral titer, n = 3 for each group. ( P ) ABR thresholds at 4 weeks of Clic5 −/ − injected with scAAV .Clic5 at different viral doses: 1.52 × 10¹³ gc/ml ( n = 10), 1.52 × 10 12 gc/ml ( n = 3) and 1.52 × 10 11 gc/ml ( n = 3). Data information: Statistical tests were two-way ANOVA with Holm–Sidak correction for multiple comparisons for ( D – F , I – N , P ), and one-way ANOVA followed by Tukey correction for multiple comparisons for ( O ). Plots show mean ± SD. ns not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (Exact P values are provided in Appendix Table S ). Scale bars = 10 μm for ( A , C , H ), 2 μm for ( B ), 0.5 μm for OHC ( G ), and 2 μm for IHC ( G ). .

Article Snippet: Imaris 3D Interactive Microscopy Visualization software , Oxford Instruments . , version 10.1.

Techniques: Injection, Staining, FLAG-tag, Electron Microscopy, Expressing