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. 2022 Oct 11;11:e77419. doi: 10.7554/eLife.77419

Figure 4. EZ Clear processed samples are compatible with wholemount immunofluorescent staining.

Figure 4.

(A) Six-step, standard wholemount immunofluorescent staining procedure for EZ Clear treated mouse brains. (B) Whole organ immunostaining of EZ Clear processed mouse brains using iDISCO or standard immunostaining (direct IHC) protocols along with To-Pro 3 staining to label nuclei (digital sectioned transversely at 2.5 mm) and (C) anti-smooth muscle α-actin conjugated to Cy3 to label smooth muscle cells and arteries (color-coded depth projection). (D) Comparison of the To-Pro 3 penetrance across the lateral axis between iDISCO and standard IHC processed samples. Quantitative comparison of mean fluorescence intensity of (E) To-Pro 3 and (F) αSMA-Cy3 at different imaging depths (dorsal to ventral) shows no significant difference between EZ Clear treated brains stained with iDISCO and standard IHC protocols (n=3, two-way ANOVA and multiple comparisons, error bars represent standard deviation [SD], ns = not significant.). (G–J) Thy1-EGFP-M mouse brain processed with EZ Clear, wholemount immunostained using a standard immunofluorescent protocol with antibodies raised against GFAP (red, glia) and wholemount imaged by lightsheet fluorescence microscopy (LSFM) at the (G and H) cortex and the intersection of the (I and J) corpus callosum (CC) and the commissure of the fornix (CoF).

Figure 4—source data 1. Measurements To-Pro 3 penetrance across the lateral axis between iDISCO and standard IHC processed samples.
Quantitative comparison of mean fluorescence intensity of To-Pro 3 and αSMA-Cy3 at different imaging depths (dorsal to ventral) between iDISCO and standard IHC processed samples.
elife-77419-fig4-data1.xlsx (590.6KB, xlsx)