Mesruh Turkekul

Machine-based method for multiplex in situ molecular characterization of tissues by immunofluorescence detection

Immunofluorescent staining is an informative tool that is widely used in basic research. Automation of immunostaining improves reproducibility and quality of the results. Up to now, use of automation in immunofluorescent staining was mostly limited to one marker. Here we present tyramide signal amplification based method of multiple marker immunofluorescent detection, including detection of antibodies, raised in the same species, in tissue sections and cultured cells. This method can be beneficial for both basic and clinical research.

Internalization of secreted antigen–targeted antibodies by the neonatal Fc receptor for precision imaging of the androgen receptor axis

A radiolabeled antibody against a secreted antigen uses Fc receptor–mediated internalization for cancer imaging and therapy. Prostate cancer hide-and-seek Prostate cancer is typically treated by targeting the androgen receptor, at least initially, but there is no convenient way to monitor the receptor’s activity or to determine when a tumor is becoming resistant to treatment. Although the androgen receptor cannot be imaged directly at this time, Thorek et al. identified an enzyme called human kallikrein-related peptidase 2 (hK2), whose activation requires signaling through the androgen receptor pathway. The authors used a radiolabeled antibody against hK2 in mouse models and human tissues to accurately detect prostate cancer lesions, including bone metastases, and to monitor their status during the course of treatment. Targeting the androgen receptor (AR) pathway prolongs survival in patients with prostate cancer, but resistance rapidly develops. Understanding this resistance is confounded by a lack of noninvasive means to assess AR activity in vivo. We report intracellular accumulation of a secreted antigen–targeted antibody (SATA) that can be used to characterize disease, guide therapy, and monitor response. AR-regulated human kallikrein-related peptidase 2 (free hK2) is a prostate tissue–specific antigen produced in prostate cancer and androgen-stimulated breast cancer cells. Fluorescent and radio conjugates of 11B6, an antibody targeting free hK2, are internalized and noninvasively report AR pathway activity in metastatic and genetically engineered models of cancer development and treatment. Uptake is mediated by a mechanism involving the neonatal Fc receptor. Humanized 11B6, which has undergone toxicological tests in nonhuman primates, has the potential to improve patient management in these cancers. Furthermore, cell-specific SATA uptake may have a broader use for molecularly guided diagnosis and therapy in other cancers.

Understanding the three-dimensional world from two-dimensional immunofluorescent adjacent sections

Visualizing tissue structures in three-dimensions (3D) is crucial to understanding normal and pathological phenomena. However, staining and imaging of thick sections and whole mount samples can be challenging. For decades, researchers have serially sectioned large tissues and painstakingly reconstructed the 3D volume. Advances in automation, from sectioning to alignment, now greatly accelerate the process. In addition, immunofluorescent staining methods allow multiple antigens to be simultaneously detected and analyzed volumetrically. The objective was to incorporate multi-channel immunofluorescent staining and automation in 3D reconstruction of serial sections for volumetric analysis. Paraffin-embedded samples were sectioned manually but were processed, stained, imaged and aligned in an automated fashion. Reconstructed stacks were quantitatively analyzed in 3D. By combining automated immunofluorescent staining and tried-and-true methods of reconstructing adjacent sections, we were able to visualize, in detail, not only the geometric structures of the sample but also the presence and interactions of multiple proteins and molecules of interest within their 3D environment. Advances in technology and software algorithms have significantly expedited the 3D reconstruction of serial sections. Automated, multi-antigen immunofluorescent staining will significantly broaden the range and complexity of scientific questions that can be answered with this methodology.

Double in situ detection of sonic hedgehog mRNA and pMAPK protein in examining the cell proliferation signaling pathway in mouse embryo.

Double in situ detection of RNA molecules and proteins in tissue sections is not trivial. A successful experiment heavily depends on the preparation of the tissue as well as the quality of the probes and antibodies. Detection of two or more molecular markers also requires reagents and experimental conditions that will preserve authenticity (accuracy) of the single staining patterns. Here, we describe in detail the protocols used to detect sonic hedgehog (Shh) mRNA by in situ hybridization and immunofluorescence staining for phosphorylated mitogen-activated protein kinase (pMAPK) in the same mouse embryonic tissue sections. In addition to protocols for manual immuno-staining, we provide data from automated machine-based staining protocols and highly recommend it to achieve strong signal and reproducible results.

Validation of Anti-Mouse PDL-1 Goat Polyclonal Antibody Staining with Mouse PDL-1 In Situ Hybridization on Adjacent Sections of Cell Pellets and Mouse Tumors

Finding a valid antibody to detect mouse programmed death ligand 1 (PDL-1) by immunohistochemistry or immunofluorescence staining has been notoriously difficult. Successful validation of an antibody requires the use of multiple detection methods with the ability to compare appropriate positive and negative controls. Here, we describe in detail the protocols used to validate a mouse-specific PDL-1 antibody used in immunohistochemistry staining with an mRNA in situ hybridization on adjacent sections of mouse B16 tumor. This validation is supported by immunohistochemistry staining of PDL-1 on B16 cell pellets either treated or not treated with IFN-gamma.

Automated Double In Situ Detection of Mouse Lgr5 mRNA and Lysozyme Protein in Examining the Neighboring Cell Types of the Mouse Intestinal Crypt

Automated detection of mRNAs and proteins in the same tissue sections is not a routine procedure. Successful experiment depends on the preparation of the tissue, the detection procedure, as well as the quality of the probes and antibodies. The multiplexed detections require experimental conditions, preserving the state of the molecular targets of interest and providing expression pattern of each target the same as in a single detection. Here we describe in detail the automated protocols used to detect mouse Lgr5 mRNA by in situ hybridization and immunofluorescence detection of lysozyme in the same mouse intestinal sections. Both the in situ hybridization and the protein detection were performed with an automated staining processor and provided strong and reproducible results.

Abstract LB-095: Distribution and clearance of single-walled carbon nanotubes in mouse tissues: in situ detection, imaging and analysis

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Single-walled carbon nanotubes (SWCNT) are the subject of expanding research in the fields of targeted drug delivery and biosensors for disease treatment and monitoring. Functionalization of the SWCNT has proven to enhance the efficiency of distribution in the organism. The goal of this project was to establish and standardize methods of detection and characterization of the local tissue distribution of functionalized SWCNT in mouse models. We investigated the distribution and clearance of SWCNT in several tissues at various times after injection, up to one month. We also try to standardize methods of tracking SWCNT in both live and fixed tissues and image analysis protocols to quantify the distribution of the SWCNT in tissues. Another aim is to evaluate local immune response to SWCNT. We have analyzed liver, kidney, spleen, lung, brain, ovary, colon and small intestine from mice sacrificed 24 hours, 3 days, 7 days and 30 days following the injection of SWCNT. Our evaluation revealed that some organs, like kidney and small intestine, retain SWCNTs for at least a month. Slow rates of SWCNT removal from these organs do not appear to affect the overall health of the animals. We have made multiple attempts to image anesthetized live animals and detect SWCNTs in situ, however, the fluorescent signal emitted from SWCNT is too low to be reliably detected. The tissues must be fixed and signals amplified through immunodetection. Detection of immune markers, especially in immunologically active tissues such as spleen is a challenge for histological experiments, but is extremely important and such experiments is underway. Our findings of SWCNT persistence in certain organs 30 days post-injection is surprising and should be studied further. This observation opens further questions about the effect of long-term presence of SWCNT in some tissues and shows that careful investigation into the advantages and disadvantages of SWCNT retention is necessary. Citation Format: Afsar Barlas, Ke Xu, Yevgeniy Romin, Simone Alidori, Dmitry Yarilin, Ning Fan, Mesruh Turkekul, Sho Fujisawa, David A. Scheinberg, Michael R. McDevitt, Katia Manova-Todorova. Distribution and clearance of single-walled carbon nanotubes in mouse tissues: in situ detection, imaging and analysis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-095. doi:10.1158/1538-7445.AM2015-LB-095

Understanding 3-Dimensional World from 2-Dimensional Immunofluorescent Adjacent Sections

In many fields of biological sciences including embryology and cancer research, understanding of 3-dimensional structures is crucial to uncovering normal and pathological phenomena. While the most optimal method would be to directly observe the complete object without any destruction, staining and imaging of thick sections and whole mount samples can be challenging. For decades, researchers have serially sectioned large tissues stained each with chromogenbased immunohistologicalmethods and painstakingly reconstructed the 3-dimensional volume. The limiting factor with immunohistological staining is the difficulty in detecting multiple antigens with different chromogens on the same tissue. At our Molecular Cytology Core Facility at Memorial Sloan-Kettering Cancer Center, we successfully and routinely perform immunofluorescent staining using automated staining machines and have combined IF staining and 3D reconstruction of serial sections.This method allows simultaneous detection of up to four different antigens on the same sections in a highly reproducible and specific manner. The resulting stack can be a stunning visualization of 3D structure and be quantitatively analyzed.