an-in-vivo-murine-sciatic-nerve-model-of-perineural-invasion

An <em>In Vivo</em> Murine Sciatic Nerve Model of Perineural Invasion

Cancer cells invade nerves through a process termed perineural invasion (PNI), in which cancer cells proliferate and migrate in the nerve ...

Memorial Sloan Kettering Cancer Center

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.

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

Radiofrequency (RF) ablation (RFA) is a minimally invasive treatment for colorectal-cancer liver metastases (CLM) in selected nonsurgical patients. Unlike surgical resection, RFA is not followed by routine pathological examination of the target tumor and the surrounding liver tissue. The aim of this study was the evaluation of apoptotic events after RFA. Specifically, we evaluated YO-PRO-1 (YP1), a green fluorescent DNA marker for cells with compromised plasma membrane, as a potential, early marker of cell death. YP1 was applied on liver tissue adherent on the RF electrode used for CLM ablation, as well as on biopsy samples from the center and the margin of the ablation zone as depicted by dynamic CT immediately after RFA. Normal pig and mouse liver tissues were used for comparison. The same samples were also immunostained for fragmented DNA (TUNEL assay) and for active mitochondria (anti-OxPhos antibody). YP1 was also used simultaneously with propidium iodine (PI) to stain mouse liver and samples from ablated CLM. Following RFA of human CLM, more than 90 % of cells were positive for YP1. In nonablated, dissected pig and mouse liver however, we found similar YP1 signals (93.1 % and 65 %, respectively). In samples of intact mouse liver parenchyma, there was a significantly smaller proportion of YP1 positive cells (22.7 %). YP1 and PI staining was similar for ablated CLM. However in dissected normal mouse liver there was initial YP1 positivity and complete absence of the PI signal and only later there was PI signal.

Evaluation of YO-PRO-1 as an early marker of apoptosis following radiofrequency ablation of colon cancer liver metastases.

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. 

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

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. 

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

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.

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.

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.

Article	Year
Double in situ detection of sonic hedgehog mRNA and pMAPK protein in examining the cell proliferation signaling pathway in mouse embryo. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 21370036	2011
Evaluation of YO-PRO-1 as an early marker of apoptosis following radiofrequency ablation of colon cancer liver metastases. Cytotechnology\| PubMed ID: 24065619	2014
Machine-based method for multiplex in situ molecular characterization of tissues by immunofluorescence detection. Scientific reports\| PubMed ID: 25826597	2015
Understanding the three-dimensional world from two-dimensional immunofluorescent adjacent sections. Journal of pathology informatics\| PubMed ID: 26110094	2015
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. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 28185198	2017
Automated Double In Situ Detection of Mouse Lgr5 mRNA and Lysozyme Protein in Examining the Neighboring Cell Types of the Mouse Intestinal Crypt. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 28185199	2017

Ning Fan

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