We evaluate the utility of our approach in antibody staining and in nanoparticle imaging, which can inform but also constitute therapeutic intervention, at both cellular and subcellular resolution. Particularly, we also used a B16 tumor line that overexpresses HER2, which we used in a previously described fully-immunocompetent syngeneic C57BL/6 model which is tolerant to the transgenic marker 13. Here we evaluate an alternative clearing approach called PACT (Passive Clarity Technique) and expand on their integrated CRISPR-based gene editing with a tissue-clearing workflow in the B16 mouse melanoma model. 9 recently applied a combined CRISPR editing of melanoma workflow in combination with a tissue expansion-based clearing termed CUBIC to a B16 mouse melanoma model, a highly melanin-rich solid tumor model 10, 11, 12, which can also be used to investigate antibody and nanoparticle targeting. To address these limitations, Tikoo et al. Although several protocols exist for removing various naturally occurring pigments using either tissue-clearing protocols or various bleaching agents and acetone in combination with tissue clearing, the results of these efforts on melanin removal have shown limited success 6, 7, 8. This limitation is most likely due to melanin’s poor solubility in both lipid and water-based solvents 5, 6 and thus its resistance to extraction. However, a major limitation of this approach is that it is not easily applicable to tissues exhibiting melanin pigmentation, as melanin is a pigment known to cause strong light absorption and scattering and thus reduction in optical imaging quality 2, 4. A central feature in these tissue-clearing methods is homogenizing the refractive index and the removal of lipids in unpigmented tissues. With CRISPR-Clear we have unprecedented access to optical interrogation in considerable portions of intact melanoma tissue for stained surface markers, expressed fluorescent proteins, of subcellular compartments, and of the vasculature.ĭriven by research on the mammalian brain, various tissue-clearing methods have been established to enable 3D large-volume optical imaging 1, 2, 3. We demonstrate the utility in detailed tumor characterization by staining for targeting antibodies and nanoparticles, as well as expressed fluorescent proteins. We demonstrate its applicability to highly melanin-rich B16-derived solid tumors, including one made transgenic for HER2, constituting one of very few syngeneic mouse tumors that can be used in immunocompetent models. To address this challenge, we have developed a CRISPR-based gene editing approach that is easily incorporated into existing tissue-clearing workflows such the PACT clearing method. In contrast, melanin-rich pigmented tissue, of great interest in the investigation of melanomas, remains challenging. Current techniques have been optimized for imaging sparsely pigmented organs such as the mammalian brain.

Tissue clearing combined with deep imaging has emerged as a powerful technology to expand classical histological techniques.

Agilent 34972a the signal path