Antibody Penetration Research Articles

Immunostaining of Whole-Mount Drosophila Testes for 3D Confocal Analysis of Large Spermatocytes.

Drosophila testes are a powerful model system for studying biological processes including stem cell biology, nuclear architecture, meiosis and sperm development. However, immunolabeling of the whole Drosophila testis is often associated with significant non-uniformity of staining due to antibody penetration. Squashed preparations only partially overcome the problem since it decreases the 3D quality of the analyses. Herein, we describe a whole-mount protocol using NP40 and heptane during fixation together with immunolabeling in liquid media. It preserves the volume suitable for confocal microscopy together with reproducible and reliable labeling. We show different examples of 3D reconstitution of spermatocyte nuclei from confocal sections. The intra- and inter-testes reproducibility allows 3D quantification and comparison of fluorescence between single cells from different genotypes. We used different components of the intranuclear MINT structure (Mad1-containing Intra Nuclear Territory) as well as two components associated with the nuclear pore complex to illustrate this protocol and its applications on the largest cells of the testis, the S4-S5 spermatocytes.

Histological-Based Stainings using Free-Floating Tissue Sections.

Immunohistochemistry is a widely used technique to visualize specific tissue structures as well as protein expression and localization. Two alternative approaches are widely used to handle the tissue sections during the staining procedure, one approach consists of mounting the sections directly on glass slides, while a second approach, the free-floating, allows for fixed sections to be maintained and stained while suspended in solution. Although slide-mounted and free-floating approaches may yield similar results, the free-floating technique allows for better antibody penetration and thus should be the method of choice when thicker sections are to be used for 3D reconstruction of the tissues, for example when the focus of the experiment is to gain information on dendritic and axonal projections in brain regions. In addition, since the sections are kept in solution, a single aliquot can easily accommodate 30 to 40 sections, handling of which is less laborious, particularly in large-scale biomedical studies. Here, we illustrate how to apply the free-floating method to fluorescent immunohistochemistry staining, with a major focus on brain sections. We will also discuss how the free-floating technique can easily be modified to fit the individual needs of researchers and adapted to other tissues as well as other histochemical-based stainings, such as hematoxylin and eosin and cresyl violet, as long as tissue samples are properly fixed, typically with paraformaldehyde or formalin.

Click Reaction-Assisted Peptide Immune Checkpoint Blockade for Solid Tumor Treatment.

One of the major challenges of immune checkpoint blockade (ICB) is the poor penetration of antibody for solid tumor treatment. Herein, peptides with deeper penetration capability are used to develop a click reaction-assisted peptide immune checkpoint blockade (CRICB) strategy that could in situ construct assemblies, enabling enhanced accumulation and prolonged PD-L1 occupancy, ultimately realizing high-performance tumor inhibition. First, the free DBCO-modified targeting peptide (TP) efficiently recognizes and binds PD-L1 in a deep solid tumor. Upon a reagent-free click reaction with a subsequently introduced azide-tethered assembled peptide (AP), the click reaction results in spontaneous self-aggregation in situ with enhanced accumulation and prolonged occupancy. In addition, the penetration of TP-AP (121.2 ± 15.5 μm) is significantly enhanced compared with that of an antibody (19.9 ± 5.6 μm) in a solid tumor tissue. More importantly, significant immunotherapy effects and negligible side effects are observed in 4T1 and CT26 tumor-bearing mice models treated with TP-AP, suggesting the high-performance tumor inhibition attributed to the CRICB strategy. In summary, this CRICB strategy manifest the preferable effects of immune checkpoint blockade, thereby extending the biomedical application of assembling peptides.

Identification of Retinal Ganglion Cells from β-III Stained Fluorescent Microscopic Images.

Optic nerve crush in mouse model is widely used for investigating the course following retinal ganglion cell (RGCs) injury. Manual cell counting from β-III tubulin stained microscopic images has been routinely performed to monitor RGCs after an optic nerve crush injury, but is time-consuming and prone to observer variability. This paper describes an automatic technique for RGC identification. We developed and validated (i) a sensitive cell candidate segmentation scheme and (ii) a classifier that removed false positives while retaining true positives. Two major contributions were made in cell candidate segmentation. First, a homomorphic filter was designed to adjust for the inhomogeneous illumination caused by uneven penetration of β-III tubulin antibody. Second, the optimal segmentation parameters for cell detection are highly image-specific. To address this issue, we introduced an offline-online parameter tuning approach. Offline tuning optimized model parameters based on training images and online tuning further optimized the parameters at the testing stage without needing access to the ground truth. In the cell identification stage, 31 geometric, statistical and textural features were extracted from each segmented cell candidate, which was subsequently classified as true or false positives by support vector machine. The homomorphic filter and the online parameter tuning approach together increased cell recall by 28%. The entire pipeline attained a recall, precision and coefficient of determination (r2) of 85.3%, 97.1% and 0.994. The availability of the proposed pipeline will allow efficient, accurate and reproducible RGC quantification required for assessing the death/survival of RGCs in disease models.

Disialoganglioside GD2 Expression in Solid Tumors and Role as a Target for Cancer Therapy.

Gangliosides are carbohydrate-containing sphingolipids that are widely expressed in normal tissues, making most subtypes unsuitable as targets for cancer therapy. However, the disialoganglioside GD2 subtype has limited expression in normal tissues but is overexpressed across a wide range of tumors. Disialoganglioside GD2 can be considered a tumor-associated antigen and well-suited as a target for cancer therapy. Disialoganglioside GD2 is implicated in tumor development and malignant phenotypes through enhanced cell proliferation, motility, migration, adhesion, and invasion, depending on the tumor type. This provides a rationale for targeting disialoganglioside GD2 in cancer therapy with the development of anti-GD2 monoclonal antibodies and other therapeutic approaches. Anti-GD2 monoclonal antibodies target GD2-expressing tumor cells, leading to phagocytosis and destruction by means of antibody-dependent cell-mediated cytotoxicity, lysis by complement-dependent cytotoxicity, and apoptosis and necrosis through direct induction of cell death. Anti-GD2 monoclonal antibodies may also prevent homing and adhesion of circulating malignant cells to the extracellular matrix. Disialoganglioside GD2 is highly expressed by almost all neuroblastomas, by most melanomas and retinoblastomas, and by many Ewing sarcomas and, to a more variable degree, by small cell lung cancer, gliomas, osteosarcomas, and soft tissue sarcomas. Successful treatment of disialoganglioside GD2-expressing tumors with anti-GD2 monoclonal antibodies is hindered by pharmacologic factors such as insufficient antibody affinity to mediate antibody-dependent cell-mediated cytotoxicity, inadequate penetration of antibody into the tumor microenvironment, and toxicity related to disialoganglioside GD2 expression by normal tissues such as peripheral sensory nerve fibers. Nonetheless, anti-GD2 monoclonal antibody dinutuximab (ch14.18) has been approved by the U.S. Food and Drug Administration and dinutuximab beta (ch14.18/CHO) has been approved by the European Medicines Agency for the treatment of high-risk neuroblastoma in pediatric patients. Clinical trials of anti-GD2 therapy are currently ongoing in patients with other types of disialoganglioside GD2-expressing tumors as well as neuroblastoma. In addition to anti-GD2 monoclonal antibodies, anti-GD2 therapeutic approaches include chimeric antigen receptor T-cell therapy, disialoganglioside GD2 vaccines, immunocytokines, immunotoxins, antibody–drug conjugates, radiolabeled antibodies, targeted nanoparticles, and T-cell engaging bispecific antibodies. Clinical trials should clarify further the potential of anti-GD2 therapy for disialoganglioside GD2-expressing malignant tumors.

TAG-72-Targeted α-Radionuclide Therapy of Ovarian Cancer Using 225Ac-Labeled DOTAylated-huCC49 Antibody.

Radioimmunotherapy, an approach using radiolabeled antibodies, has had minimal success in the clinic with several β-emitting radionuclides for the treatment of ovarian cancer. Alternatively, radioimmunotherapy with α-emitters offers the advantage of depositing much higher energy over shorter distances but was thought to be inappropriate for the treatment of solid tumors, for which antibody penetration is limited to a few cell diameters around the vascular system. However, the deposition of high-energy α-emitters to tumor markers adjacent to a typical leaky tumor vascular system may have large antitumor effects at the tumor vascular level, and their reduced penetration in normal tissue would be expected to lower off-target toxicity. Methods: To evaluate this concept, DOTAylated-huCC49 was labeled with the α-emitter 225Ac to target tumor-associated glycoprotein 72-positive xenografts in a murine model of ovarian cancer. Results:225Ac-labeled DOTAylated-huCC49 radioimmunotherapy significantly reduced tumor growth in a dose-dependent manner (1.85, 3.7, and 7.4 kBq), with the 7.4-kBq dose extending survival by more than 3-fold compared with the untreated control. Additionally, a multitreatment regime (1.85 kBq followed by 5 weekly doses of 0.70 kBq for a total of 5.4 kBq) extended survival almost 3-fold compared with the untreated control group, without significant off-target toxicity. Conclusion: These results establish the potential for antibody-targeted α-radionuclide therapy for ovarian cancer, which may be generalized to α-radioimmunotherapy in other solid tumors.

A Viral Toolbox of Genetically Encoded Fluorescent Synaptic Tags.

SummaryFibronectin intrabodies generated with mRNA display (FingRs) are a recently developed tool for labeling excitatory or inhibitory synapses, with the benefit of not altering endogenous synaptic protein expression levels or synaptic transmission. Here, we generated a viral vector FingR toolbox that allows for multi-color, neuron-type-specific labeling of excitatory or inhibitory synapses in multiple brain regions. We screened various fluorophores, FingR fusion configurations, and transcriptional control regulations in adeno-associated virus (AAV) and retrovirus vector designs. We report the development of a red FingR variant and demonstrated dual labeling of excitatory and inhibitory synapses in the same cells. Furthermore, we developed cre-inducible FingR AAV variants and demonstrated their utility, finding that the density of inhibitory synapses in aspiny striatal cholinergic interneurons remained unchanged in response to dopamine depletion. Finally, we generated FingR retroviral vectors, which enabled us to track the development of excitatory and inhibitory synapses in hippocampal adult-born granule cells.

Increased Tumor Penetration of Single-Domain Antibody-Drug Conjugates Improves In Vivo Efficacy in Prostate Cancer Models.

Targeted delivery of chemotherapeutics aims to increase efficacy and lower toxicity by concentrating drugs at the site-of-action, a method embodied by the seven current FDA-approved antibody-drug conjugates (ADC). However, a variety of pharmacokinetic challenges result in relatively narrow therapeutic windows for these agents, hampering the development of new drugs. Here, we use a series of prostate-specific membrane antigen-binding single-domain (Humabody) ADC constructs to demonstrate that tissue penetration of protein-drug conjugates plays a major role in therapeutic efficacy. Counterintuitively, a construct with lower in vitro potency resulted in higher in vivo efficacy than other protein-drug conjugates. Biodistribution data, tumor histology images, spheroid experiments, in vivo single-cell measurements, and computational results demonstrate that a smaller size and slower internalization rate enabled higher tissue penetration and more cell killing. The results also illustrate the benefits of linking an albumin-binding domain to the single-domain ADCs. A construct lacking an albumin-binding domain was rapidly cleared, leading to lower tumor uptake (%ID/g) and decreased in vivo efficacy. In conclusion, these results provide evidence that reaching the maximum number of cells with a lethal payload dose correlates more strongly with in vivo efficacy than total tumor uptake or in vitro potency alone for these protein-drug conjugates. Computational modeling and protein engineering can be used to custom design an optimal framework for controlling internalization, clearance, and tissue penetration to maximize cell killing. SIGNIFICANCE: A mechanistic study of protein-drug conjugates demonstrates that a lower potency compound is more effective in vivo than other agents with equal tumor uptake due to improved tissue penetration and cellular distribution.

Whole-mount Immunohistochemistry of Adult Zebrafish Retina for Advanced Imaging.

Immunohistochemistry is a widely used technique to examine the expression and subcellular localization of proteins. This technique relies on the specificity of antibodies and requires adequate penetration of antibodies into tissues. The latter is especially challenging for thick specimens, such as embryos and other whole-mount preparations. Here we describe an improved method of immunohistochemistry for retinal whole-mount preparations. We report that a cocktail of three reagents, Triton X-100, Tween-20, and DMSO, in blocking and antibody dilution buffers strongly enhances immunolabeling in whole-mount retinas from adult zebrafish. In addition, we establish that in whole retinal tissues, a classic epitope retrieval method, based on citrate buffer, is effective for immunolabeling membrane-associated proteins. Overall, this simple modification allows precise and reproducible immunolabeling of proteins in retinal whole-mounts.

Intracellular delivery of therapeutic antibodies into specific cells using antibody-peptide fusions

Because of their favorable properties as macromolecular drugs, antibodies are a very successful therapeutic modality for interfering with disease-relevant targets in the extracellular space or at the cell membrane. However, a large number of diseases involve cytosolic targets and designing antibodies able to efficiently reach intracellular compartments would expand the antibody-tractable conditions. Here, we genetically fused cell penetrating peptides (CPPs) at various positions to an antibody targeting cancer cells, evaluated the developability features of the resulting antibody-peptide fusions and the ability of selected constructs to reach the cytosol. We first determined positions in the IgG structure that were permissive to CPP incorporation without destabilizing the antibody. Fusing CPPs to the C-terminus of the light chain and either before or after the hinge had the least effect on antibody developability features. These constructs were further evaluated for cell penetration efficiency. Two out of five tested CPPs significantly enhanced antibody penetration into the cytosol, in particular when fused before or after the hinge. Finally, we demonstrate that specific antibody binding to the cell surface target is necessary for efficient cell penetration of the CPP-antibody fusions. This study provides a solid basis for further exploration of therapeutic antibodies for intracellular targets.

Phase 1 Trial of Human IL-15 (rhIL-15) and Obinutuzumab for Relapsed and Refractory Chronic Lymphocytic Leukemia

Background: Of the several drugs and drug combinations approved for treatment of relapsed and refractory chronic lymphocytic leukemia (CLL), the reported complete response rates are no greater than 30%. Obinutuzumab is a glycoengineered, humanized type 2 anti-CD20 monoclonal antibody thought to engage the immune system by directly activating antibody- dependent, cell-mediated cytotoxicity (ADCC); it is approved for treatment of chronic lymphocytic leukemia in combination with chlorambucil. The key mediators of ADCC are polymorphonuclear neutrophils, monocytes, and natural killer (NK) cells. Recombinant human Interleukin-15 (rhIL-15) is a stimulatory cytokine that promotes the differentiation and activation of NK cells, monocytes, and long-term CD8+ memory T-cells. In a Phase I trial, administration of rhIL-15 as a 5-day continuous intravenous infusion (civ) was associated with up to 45-fold increase in the number of NK cells at well- tolerated dose levels. Preclinical murine lymphoid malignancy models have shown increased efficacy of monoclonal antibodies when administered together with rhIL-15; BL/6 mice inoculated with EL4-CD20 cells (a syngeneic lymphoma line); including significant prolongation of survival with the IL-15/Rituximab combination compared to either drug given as single agent (90% v. 30% alive at 75 days). We hypothesized that rhIL-15-associated increase in NK cell number and activity would improve efficacy of obinutuzumab in treatment of relapsed and refractory CLL, and would increase the duration and depth of response; this phase I trial is testing the safety of the combination. Primary objective: determine the safety, toxicity profile, dose-limiting toxicity (DLT) and the maximum tolerated dose (MTD) of civ rhIL-15 administration in combination with obinutuzumab treatment Secondary objectives: 1) evaluate the potential antitumor activity of the combination of rhIL-15 and obinutuzumab by assessing the clinical response rate, minimal residual disease (MRD) status, progression-free survival, and overall survival in patients with relapsed and refractory CLL; 2) define the effects of rhIL-15 on the ADCC mediated by obinutuzumab using ex vivo peripheral blood mononuclear cells (PBMCs); 3) characterize the biological effects of rhIL-15 administered with obinutuzumab on the percentages and absolute numbers of circulating lymphocytes (T and NK cells) and the T- cell subsets (including naïve, central, and effector memory subsets) by flow cytometry Exploratory objectives: identify biomarkers predictive of response to rhIL-15 and obinutuzumab treatment, such as circulating tumor DNA (ctDNA) and baseline cytokine levels Eligibility criteria: 1) age ≥ 18 years; 2) ECOG ≤ 1; 3) Diagnosis of CLL or small lymphocytic lymphoma (SLL) with ≥ 50% of B cells expressing CD20; 4) measurable or evaluable disease that is refractory or relapsed following therapy with a BTK inhibitor OR have relapsed/refractory CLL and are intolerant to BTK inhibitor therapy; patients with del(17p) must also be refractory or relapsed after, or intolerant to, therapy with venetoclax; 5) adequate organ function parameters as defined within the protocol; 6) active disease requiring treatment, as defined within the protocol. Study design: a single institution non-randomized phase I dose escalation study evaluating increasing doses of civ rhIL-15 in combination with obinutuzumab using a 3 + 3 dose escalation design. On days 1-5 of each 4-week cycle, rhIL-15 will be administered by civ at dose levels 0.5, 1, and 2 mcg/kg/day. During the first cycle, obinutuzumab will be administered at a dose of 100 mg by IV on day 4, 900 mg on day 5, 1,000 mg on day 11, and 1,000 mg on day 18; then 1,000 mg on day 4 of each subsequent cycle. Infusion reaction, antimicrobial, and tumor lysis syndrome prophylaxis will be administered per manufacturer's recommendations. Treatment will continue up to 6 cycles, or until unacceptable toxicity or progressive disease. Up to 24 patients will be enrolled in the study. Correlative studies: 1) optional lymph node biopsy at baseline and after the first week of treatment (C1D8) for tissue immune cell subsets and quantifying antibody penetration; 2) lymphocyte subset testing, ctDNA, and cell and plasma banking on days 1, 4, 8, and 11 of cycle 1, and days 1 and 8 of cycles 2-6; 3) ADCC capacity of ex vivo NK cells on C1D1, 4, and 8. One patient has started treatment to date. Enrollment is ongoing. Figure Disclosures Wiestner: Acerta: Research Funding; Pharmayclics: Research Funding; Merck: Research Funding; Nurix: Research Funding. Waldmann:Bioniz: Membership on an entity's Board of Directors or advisory committees.

Brief freezing steps lead to robust immunofluorescence in the Drosophila nervous system.

Drosophila melanogaster possesses a complex nervous system, regulating sophisticated behavioral outputs, that serves as a powerful model for dissecting molecular mechanisms underlying neuronal function and neurodegenerative disease. Immunofluorescence techniques provide a way to visualize the spatiotemporal organization of these networks, permitting observation of their development, functional location, remodeling and, eventually, degradation. However, basic immunostaining techniques do not always result in efficient antibody penetration through the brain, and supplemental techniques to enhance permeability can compromise structural integrity, altering spatial organization. Here, slow freezing of brains is shown to facilitate antibody permeability without loss of antibody specificity or brain integrity. To demonstrate the advantages of this freezing technique, the results of two commonly used permeation methods – detergent-based and partial proteolytic digestion – are compared.

An Overview of the Current ADC Discovery Landscape.

The prototypical ADC mechanism involving antigen-mediated uptake and lysosomal release is both elegantly simple and scientifically compelling. However, recent clinical-stage failures have prompted a reevaluation of this delivery paradigm and have resulted in an array of new technologies that have the potential to improve the safety and efficacy of up and coming programs. These innovations can generally be categorized into seven areas that will be elaborated on in this chapter: (1) Exploiting new payload mechanisms; (2) Increasing the drug-antibody ratio (DAR); (3) Increasing the antibody penetration; (4) Overcoming ADC resistance mechanisms; (5) Increasing the efficiency of ADC uptake and processing; (6) Mitigating off-target payload exposure; and (7) Employment of noncytotoxic payloads. It is our belief that these seven areas capture the current "landscape" of innovations that are taking place in the design of next-generation ADCs. Together, these advancements are reshaping the ADC field and providing a path forward in the face of the recent clinical setbacks.

Optical Clearing and Imaging of Immunolabeled Kidney Tissue.

Optical clearing techniques render tissue transparent by equilibrating the refractive index throughout a sample for subsequent three-dimensional (3-D) imaging. They have received great attention in all research areas for the potential to analyze microscopic multicellular structures that extend over macroscopic distances. Given that kidney tubules, vasculature, nerves, and glomeruli extend in many directions, which have been only partially captured by traditional two-dimensional techniques so far, tissue clearing also opened up many new areas of kidney research. The list of optical clearing methods is rapidly growing, but it remains difficult for beginners in this field to choose the best method for a given research question. Provided here is a simple method that combines antibody labeling of thick mouse kidney slices; optical clearing with cheap, non-toxic and ready-to-use chemical ethyl cinnamate; and confocal imaging. This protocol describes how to perfuse kidneys and use an antigen-retrieval step to increase antibody- binding without requiring specialized equipment. Its application is presented in imaging different multicellular structures within the kidney, and how to troubleshoot poor antibody penetration into tissue is addressed. We also discuss the potential difficulties of imaging endogenous fluorophores and acquiring very large samples and how to overcome them. This simple protocol provides an easy-to-setup and comprehensive tool to study tissue in three dimensions.

Abstract LB-219: Derivation of mathematical modeling for the diffusion and penetration of monoclonal antibody delivery into tumor

Abstract Background: Commonly malignant solid tumor have an abundant tumor-associated cellular matrix (ECM). Although radioimmunothrepy (RIT) have proven to be effective and precise treatment in cancer, yet RIT has a limited performance in solid tumor, due to highly distributed ECM inside solid tumor tissue. Previously, we designed the combination RIT to enhance the penetration of monoclonal antibody (mAb), where we investigated that, using Relaxin which contain oncolytic adenovirus (OA) as combination therapy decreases tumor associated ECM. In this study, we developed mathematical modeling to explain and describe the diffusion and penetration of mAb inside the tumor with ECM. To obtain the model parameters in our developed mathematical model, we performed related experiments such as immunofluorescence and qRT-PCR. Methods: We developed the reaction-diffusion partial differential equations (PDE) for describing the dependency of cancer cell, ECM density, diffusion and penetration of mAb and ECM modulated therapy that include our experimental data, also to overcome the classical limitation of PDE method. Model included the rapid decrease of ECM by OA, and density dependent diffusion coefficient. Initial condition for solving the PDE was derived using our experimental result. Diffusion coefficient for mAb and OA was modelled for simulating actual behavior of mAb and OA. Various condition of ECM density, and structure were modelled that derives the actual result of distribution of collagen. To confirm the mathematical prediction, we designed in vitro experiment with same condition as used as “in model”. Results: Using our developed model, we successfully described relationship between ECM and mAb transport by OA, and investigated the change of ECM. Diffusion and penetration of mAb and OA were well described from tumor vessel and tumor surface. Regardless of ECM structure, all ECM structure were degraded after spreading of OA. In addition, our model simulation results predicted that mAb uptake dramatically increased in combination approach and was more than mAb alone treatment. Our developed mathematical model predicted the optimal schedule of OA pretreatment and it shows great results more than mAb pretreatment. Pretreatment results with OA shows a great anti-tumor effect more than mAb pretreatment in experimental data. Our model could well predicted and matched the experimental results. Collectively, in our study, we suggested new theoretical mathematical model framework to explain various ECM condition with drug transport Citation Format: Hyeongi Kim, Yangjin Kim, Yong-Jin Lee, Jin-su Kim. Derivation of mathematical modeling for the diffusion and penetration of monoclonal antibody delivery into tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-219.

Immunostaining in whole-mount lipid-cleared peripheral nerves and dorsal root ganglia after neuropathy in mice

Immunohistochemical characterization of primary afferent fibers (intact or after nerve damage) is traditionally performed in thin sections from dorsal root ganglia (DRGs) or in teased fibers, as light scattering in whole-mounts compromises visualization. These procedures are time-consuming, require specific equipment and advanced experimental skills. Lipid-clearing techniques are increasing in popularity, but they have never been used for the peripheral nervous system. We established a modified, inexpensive clearing method based on lipid-removal protocols to make transparent peripheral nerve tissue (inCLARITY). We compared retrograde-labeling and free-floating immunostaining with cryo-sections. Confocal microscopy on whole-mount transparent DRGs showed neurons marked with retrograde tracers applied to experimental neuromas (Retrobeads, Fluoro-ruby, Fluoro-emerald, DiI, and Fluoro-gold). After immunostaining with calcitonin gene-related peptide (peptidergic) or isolectin IB4 (non-peptidergic), nociceptors were visualized. Immunostaining in transparent whole-mount nerves allows simultaneous evaluation of the axotomized branches containing the neuroma and neighboring intact branches as they can be mounted preserving their anatomical disposition and fiber integrity. The goal of our study was to optimize CLARITY for its application in peripheral nerve tissues. The protocol is compatible with the use of retrograde tracers and improves immunostaining outcomes when compared to classical cryo-sectioning, as lack of lipids maximizes antibody penetration within the tissue.

Protein Chemical Synthesis Combined with Mirror-Image Phage Display Yields D-Peptide EGF Ligands that Block the EGF-EGFR Interaction

EGFR, the receptor for epidermal growth factor (EGF), is arguably considered the most important therapeutic target in contemporary anticancer treatments. However, 20 years after the introduction of anti-EGFR drugs into the clinical setting, a number of severe drawbacks have been observed, namely low tumour penetration of monoclonal antibodies, acquisition of resistance to tyrosine-kinase inhibitors, and complete lack of efficacy in several types of cancer. Here, we report the design of a strategy to obtain all-D peptides directed to EGF instead of EGFR and we demonstrate their ability to block efficiently the EGF-EGFR interaction. We report an efficient chemical synthesis of the enantiomeric version of human EGF. Combined with the use of mirror-image phage display, this strategy has allowed us to discovery several protease-resistant peptides able to bind to EGF. Using alpha-screen technology we have demonstrated the capacity of the best peptide candidates to disrupt the EGF-EGFR interaction.

Immunofluorescence Tomography: High-resolution 3-D reconstruction by serial-sectioning of methacrylate embedded tissues and alignment of 2-D immunofluorescence images

Immunofluorescence tomography is a high-resolution 3-D reconstruction method based on methacrylate embedding and serial-sectioning, where 2-D images of immuno-stained serial-sections are computationally aligned into image stacks, and the 3-D volume rendered. Butyl-Methyl Methacrylate (BMMA) plastic was adopted as it preserves excellent tissue morphology and can be de-plasticized easily using an organic solvent, which enables immuno-staining of serial-sections without antibody penetration issues over millimeters of 3-D reconstructed tissue (Z-depth). High axial Z-resolution over a large volume was achieved by cutting serial-sections at 2 µm thickness. Stained sections were imaged by multiple modalities, including immunofluorescence, electron microscopy and second harmonic generation (SHG), and there are advantages over confocal microscopy as the tissue does not need to be cleared, while antibody penetration or light scattering issues are minimized. The plastic serial-sections can be re-probed, without a loss in tissue structure, using low pH glycine hydrochloride antibody elution. It is a cost-effective approach as the microscopes needed are significantly cheaper than confocal microscopes and sections can be kept indefinitely. Therefore, immunofluorescence tomography is a powerful new tool to quantify sub-populations of cells in high-resolution 3-D using antibody fluorescence. This article describes the immunofluorescence tomography method for 3-D reconstruction of epithelial tissues such as mammary gland, cornea and the hair follicle.

A "Clearer" View of Pancreatic Pathology: A Review of Tissue Clearing and Advanced Microscopy Techniques.

Although pathologic lesions in the pancreas are 3-dimensional (3D) complex structures, we currently use thin 2D hematoxylin and eosin stained slides to study and diagnose pancreatic pathology. Two technologies, tissue clearing and advanced microscopy, have recently converged, and when used together they open the remarkable world of 3D anatomy and pathology to pathologists. Advances in tissue clearing and antibody penetration now make even dense fibrotic tissues amenable to clearing, and light sheet and confocal microscopies allow labeled cells deep within these cleared tissues to be visualized. Clearing techniques can be categorized as solvent-based or aqueous-based techniques, but both clearing methods consist of 4 fundamental steps, including pretreatment of specimens, permeabilization and/or removal of lipid, immunolabeling with antibody penetration, and clearing by refractive index matching. Specialized microscopes, including the light sheet microscope, the 2-photon microscope, and the confocal microscope, can then be used to visualize and evaluate the 3D histology. Both endocrine and exocrine pancreas pathology can then be visualized. The application of labeling and clearing to surgically resected human pancreatic parenchyma can provide detailed visualization of the complexities of normal pancreatic anatomy. It also can be used to characterize the 3D architecture of disease processes ranging from precursor lesions, such as pancreatic intraepithelial neoplasia lesions and intraductal papillary mucinous neoplasms, to infiltrating pancreatic ductal adenocarcinomas. The evaluation of 3D histopathology, including pathology of the pancreatic lesions, will provide new insights into lesions that previously were seen, and thought of, only in 2 dimensions.

Looking through Brains with Fast Passive CLARITY: Zebrafish, Rodents, Non-human Primates and Humans.

Recently developed CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/Immunostaining/In situ-hybridization-compatible Tis-sue-hYdrogel) technique renders the tissue transparent by removing lipids in the tissue, while preserving and stabilizing the cellular and subcellular structures. This provides effective penetration of diverse labeling probes, from primary and secondary antibodies to complementary DNA and RNA strands. Followed by high-resolution 3D imaging of neuronal cells and their projections in thick sections, tissue blocks, whole brains, or whole animals, CLARITY allows for superior quantitative analysis of neuronal tissue. Here, we provide our detailed protocol for PACT (Passive Clarity Technique) in brain tissue of diverse species, including human, non-human primate, rodents, and zebrafish. We describe the six principal steps: (1) Tissue fixation and preparation, (2) Passive lipid removal, (3) Immuno-labeling, (4) Optical clearing, (5) Imaging, (6) 3D visualization and quantification.