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Methods in Molecular Biology (Clifton,... 2019Immunofluorescence (IF) is an important immunochemical technique that allows for detection and localization of a wide variety of antigens in different types of tissues... (Review)
Review
Immunofluorescence (IF) is an important immunochemical technique that allows for detection and localization of a wide variety of antigens in different types of tissues of various cell preparations. IF allows for excellent sensitivity and amplification of signal in comparison to immunohistochemistry, employing various microscopy techniques. There are two methods available, depending on the scope of the experiment or the specific antibodies in use: direct (primary) or indirect (secondary). Here, we describe preparation of specimens preserved in different types of media and step-by-step methods for both direct and indirect immunofluorescence staining.
Topics: Antibodies; Antigens; Fluorescent Antibody Technique; Fluorescent Antibody Technique, Indirect; Humans; Immunohistochemistry; Staining and Labeling
PubMed: 30539454
DOI: 10.1007/978-1-4939-8935-5_26 -
Methods in Molecular Biology (Clifton,... 2019Immunohistochemistry (IHC) is a powerful technique that exploits the specific binding between an antibody and antigen to detect and localize specific antigens in cells... (Review)
Review
Immunohistochemistry (IHC) is a powerful technique that exploits the specific binding between an antibody and antigen to detect and localize specific antigens in cells and tissue, most commonly detected and examined with the light microscope. A standard tool in many fields in the research setting, IHC has become an essential ancillary technique in clinical diagnostics in anatomic pathology (Lin F, Chen Z. Arch Pathol Lab Med 138:1564-1577, 2014) with the advent of antigen retrieval methods allowing it to be performed conveniently on formalin fixed paraffin embedded (FFPE) tissue (Taylor CR, Shi S-R, Barr NJ. Techniques of immunohistochemistry: principles, pitfalls, and standardization. In: Dabbs DJ (ed) Diagnostic immunohistochemistry: theranostic and genomic applications, 3rd edn. Saunders, Philadelphia, 2010; Shi SR, Key ME, Kalra KL. J Histochem Cytochem 39:741-748, 1991) and automated methods for high volume processing with reproducibility (Prichard J, Hicks D, Hammond E. Automated immunohistochemistry overview. In: Fan L, Jeffrey P (eds) Handbook of practical immunohistochemistry: frequently asked questions, 2nd edn. Springer, New York, 2015). IHC is frequently utilized to assist in the classification of neoplasms, determination of a metastatic tumor's site of origin and detection of tiny foci of tumor cells inconspicuous on routine hematoxylin and eosin (H&E) staining. Furthermore, it is increasingly being used to provide predictive and prognostic information, such as in testing for HER2 amplification in breast cancer (Wolff AC, Hammond MEH, Hicks DG et al. Arch Pathol Lab Med 138:241-256, 2014) in addition to serving as surrogate markers for molecular alterations in neoplasms, including IDH1 and ATRX mutations in brain tumors (Appin CL, Brat DJ. Mol Aspects Med. 45:87-96, 2015). In this chapter we describe the basic methods of immunohistochemical staining which has become an essential tool in the daily practice of anatomic pathology worldwide.
Topics: Biomarkers, Tumor; Female; Humans; Immunohistochemistry; Paraffin Embedding; Prognosis; Tissue Fixation
PubMed: 30539453
DOI: 10.1007/978-1-4939-8935-5_25 -
Cancer Communications (London, England) Apr 2020Conventional immunohistochemistry (IHC) is a widely used diagnostic technique in tissue pathology. However, this technique is associated with a number of limitations,... (Review)
Review
Conventional immunohistochemistry (IHC) is a widely used diagnostic technique in tissue pathology. However, this technique is associated with a number of limitations, including high inter-observer variability and the capacity to label only one marker per tissue section. This review details various highly multiplexed techniques that have emerged to circumvent these constraints, allowing simultaneous detection of multiple markers on a single tissue section and the comprehensive study of cell composition, cellular functional and cell-cell interactions. Among these techniques, multiplex Immunohistochemistry/Immunofluorescence (mIHC/IF) has emerged to be particularly promising. mIHC/IF provides high-throughput multiplex staining and standardized quantitative analysis for highly reproducible, efficient and cost-effective tissue studies. This technique has immediate potential for translational research and clinical practice, particularly in the era of cancer immunotherapy.
Topics: Fluorescent Antibody Technique; Humans; Immunohistochemistry; Immunotherapy; Neoplasms
PubMed: 32301585
DOI: 10.1002/cac2.12023 -
The Oncologist May 2018Chemotherapy has been the historical mainstay of treatment for patients with breast cancer, with immunohistochemical markers and tumor characteristics driving treatment... (Review)
Review
PURPOSE
Chemotherapy has been the historical mainstay of treatment for patients with breast cancer, with immunohistochemical markers and tumor characteristics driving treatment decisions. The discovery of different intrinsic subtypes of breast cancer has advanced the understanding of breast cancer, with gene-based assays shedding further light on tumor behavior and response to treatment.
DESIGN
This review focuses on the landscape of the luminal A subtype, its definition based on immunohistochemistry (IHC) and gene assays, the prognostic and predictive value of these assays, guideline recommendations, and treatment implications.
RESULTS
Clinical studies of the prognostic value of gene-based and IHC-based assays in patients with luminal A-subtype breast cancers suggest a better prognosis for these patients compared with those with breast cancers of other subtypes.
CONCLUSION
In today's era of precision medicine, the best treatment regimen for patients with luminal A-subtype tumors is still undetermined, but available data raise the question whether chemotherapy can be omitted and endocrine therapy alone is sufficient for this patient population.
IMPLICATIONS FOR PRACTICE
Immunohistochemical markers have traditionally guided treatment decisions in breast cancer. However, advances in gene-expression profiling and availability of gene-based assays have launched these newer tests into everyday clinical practice. Luminal A-subtype tumors are a unique subset that may have favorable tumor biology. Properly defining this tumor subtype is important and may identify a subset of patients for whom endocrine therapy alone is sufficient.
Topics: Breast Neoplasms; Female; Humans; Immunohistochemistry
PubMed: 29472313
DOI: 10.1634/theoncologist.2017-0535 -
International Journal of Molecular... Feb 2023Traditional immunohistochemistry (IHC) has already become an essential method of diagnosis and therapy in cancer management. However, this antibody-based technique is... (Review)
Review
Traditional immunohistochemistry (IHC) has already become an essential method of diagnosis and therapy in cancer management. However, this antibody-based technique is limited to detecting a single marker per tissue section. Since immunotherapy has revolutionized the antineoplastic therapy, developing new immunohistochemistry strategies to detect multiple markers simultaneously to better understand tumor environment and predict or assess response to immunotherapy is necessary and urgent. Multiplex immunohistochemistry (mIHC)/multiplex immunofluorescence (mIF), such as multiplex chromogenic IHC and multiplex fluorescent immunohistochemistry (mfIHC), is a new and emerging technology to label multiple biomarkers in a single pathological section. The mfIHC shows a higher performance in cancer immunotherapy. This review summarizes the technologies, which are applied for mfIHC, and discusses how they are employed for immunotherapy research.
Topics: Humans; Fluorescent Antibody Technique; Immunohistochemistry; Neoplasms; Biomarkers; Immunotherapy; Biomarkers, Tumor
PubMed: 36834500
DOI: 10.3390/ijms24043086 -
American Journal of Physiology. Cell... Apr 2011Fluorescence microscopy is one of the most powerful tools for elucidating the cellular functions of proteins and other molecules. In many cases, the function of a...
Fluorescence microscopy is one of the most powerful tools for elucidating the cellular functions of proteins and other molecules. In many cases, the function of a molecule can be inferred from its association with specific intracellular compartments or molecular complexes, which is typically determined by comparing the distribution of a fluorescently labeled version of the molecule with that of a second, complementarily labeled probe. Although arguably the most common application of fluorescence microscopy in biomedical research, studies evaluating the "colocalization" of two probes are seldom quantified, despite a diversity of image analysis tools that have been specifically developed for that purpose. Here we provide a guide to analyzing colocalization in cell biology studies, emphasizing practical application of quantitative tools that are now widely available in commercial and free image analysis software.
Topics: Biology; Fluorescent Dyes; Image Interpretation, Computer-Assisted; Immunohistochemistry; Microscopy, Fluorescence; Software
PubMed: 21209361
DOI: 10.1152/ajpcell.00462.2010 -
PloS One 2015In situ hybridization (ISH) is an extremely useful tool for localizing gene expression and changes in expression to specific cell populations in tissue samples across...
In situ hybridization (ISH) is an extremely useful tool for localizing gene expression and changes in expression to specific cell populations in tissue samples across numerous research fields. Typically, a research group will put forth significant effort to design, generate, validate and then utilize in situ probes in thin or ultrathin paraffin embedded tissue sections. While combining ISH and IHC is an established technique, the combination of RNAscope ISH, a commercially available ISH assay with single transcript sensitivity, and IHC in thick free-floating tissue sections has not been described. Here, we provide a protocol that combines RNAscope ISH with IHC in thick free-floating tissue sections from the brain and allows simultaneous co-localization of genes and proteins in individual cells. This approach works well with a number of ISH probes (e.g. small proline-rich repeat 1a, βIII-tubulin, tau, and β-actin) and IHC antibody stains (e.g. tyrosine hydroxylase, βIII-tubulin, NeuN, and glial fibrillary acidic protein) in rat brain sections. In addition, we provide examples of combining ISH-IHC dual staining in primary neuron cultures and double-ISH labeling in thick free-floating tissue sections from the brain. Finally, we highlight the ability of RNAscope to detect ectopic DNA in neurons transduced with viral vectors. RNAscope ISH is a commercially available technology that utilizes a branched or "tree" in situ method to obtain ultrasensitive, single transcript detection. Immunohistochemistry is a tried and true method for identifying specific protein in cell populations. The combination of a sensitive and versatile oligonucleotide detection method with an established and versatile protein assay is a significant advancement in studies using free-floating tissue sections.
Topics: Animals; Brain; Immunohistochemistry; In Situ Hybridization; Male; Neurons; Primary Cell Culture; Rats
PubMed: 25794171
DOI: 10.1371/journal.pone.0120120 -
Journal of Visualized Experiments : JoVE Jun 2018Microglia are brain phagocytes that participate in brain homeostasis and continuously survey their environment for dysfunction, injury, and disease. As the first...
Microglia are brain phagocytes that participate in brain homeostasis and continuously survey their environment for dysfunction, injury, and disease. As the first responders, microglia have important functions to mitigate neuron and glia dysfunction, and in this process, they undergo a broad range of morphologic changes. Microglia morphologies can be categorized descriptively or, alternatively, can be quantified as a continuous variable for parameters such as cell ramification, complexity, and shape. While methods for quantifying microglia are applied to single cells, few techniques apply to multiple microglia in an entire photomicrograph. The purpose of this method is to quantify multiple and single cells using readily available ImageJ protocols. This protocol is a summary of the steps and ImageJ plugins recommended to convert fluorescence and bright-field photomicrographs into representative binary and skeletonized images and to analyze them using software plugins AnalyzeSkeleton (2D/3D) and FracLac for morphology data collection. The outputs of these plugins summarize cell morphology in terms of process endpoints, junctions, and length as well as complexity, cell shape, and size descriptors. The skeleton analysis protocol described herein is well suited for a regional analysis of multiple microglia within an entire photomicrograph or region of interest (ROI) whereas FracLac provides a complementary individual cell analysis. Combined, the protocol provides an objective, sensitive, and comprehensive assessment tool that can be used to stratify between diverse microglia morphologies present in the healthy and injured brain.
Topics: Animals; Immunohistochemistry; Mice; Microglia; Photomicrography; Rats
PubMed: 29939190
DOI: 10.3791/57648 -
Advances in Anatomic Pathology May 2020Immunohistochemistry represents an indispensable complement to an epidemiology and morphology-driven approach to tumor diagnosis and site of origin assignment. This... (Review)
Review
Immunohistochemistry represents an indispensable complement to an epidemiology and morphology-driven approach to tumor diagnosis and site of origin assignment. This review reflects the state of my current practice, based on 15-years' experience in Pathology and a deep-dive into the literature, always striving to be better equipped to answer the age old questions, "What is it, and where is it from?" The tables and figures in this manuscript are the ones I "pull up on the computer" when I am teaching at the microscope and turn to myself when I am (frequently) stuck. This field is so exciting because I firmly believe that, through the application of next-generation immunohistochemistry, we can provide better answers than ever before. Specific topics covered in this review include (1) broad tumor classification and associated screening markers; (2) the role of cancer epidemiology in determining pretest probability; (3) broad-spectrum epithelial markers; (4) noncanonical expression of broad tumor class screening markers; (5) a morphologic pattern-based approach to poorly to undifferentiated malignant neoplasms; (6) a morphologic and immunohistochemical approach to define 4 main carcinoma types; (7) CK7/CK20 coordinate expression; (8) added value of semiquantitative immunohistochemical stain assessment; algorithmic immunohistochemical approaches to (9) "garden variety" adenocarcinomas presenting in the liver, (10) large polygonal cell adenocarcinomas, (11) the distinction of primary surface ovarian epithelial tumors with mucinous features from metastasis, (12) tumors presenting at alternative anatomic sites, (13) squamous cell carcinoma versus urothelial carcinoma, and neuroendocrine neoplasms, including (14) the distinction of pheochromocytoma/paraganglioma from well-differentiated neuroendocrine tumor, site of origin assignment in (15) well-differentiated neuroendocrine tumor and (16) poorly differentiated neuroendocrine carcinoma, and (17) the distinction of well-differentiated neuroendocrine tumor G3 from poorly differentiated neuroendocrine carcinoma; it concludes with (18) a discussion of diagnostic considerations in the broad-spectrum keratin/CD45/S-100-"triple-negative" neoplasm.
Topics: Algorithms; Biomarkers, Tumor; Humans; Immunohistochemistry; Neoplasms
PubMed: 32205473
DOI: 10.1097/PAP.0000000000000256 -
Journal For Immunotherapy of Cancer May 2020The interaction between the immune system and tumor cells is an important feature for the prognosis and treatment of cancer. Multiplex immunohistochemistry (mIHC) and...
OBJECTIVES
The interaction between the immune system and tumor cells is an important feature for the prognosis and treatment of cancer. Multiplex immunohistochemistry (mIHC) and multiplex immunofluorescence (mIF) analyses are emerging technologies that can be used to help quantify immune cell subsets, their functional state, and their spatial arrangement within the tumor microenvironment.
METHODS
The Society for Immunotherapy of Cancer (SITC) convened a task force of pathologists and laboratory leaders from academic centers as well as experts from pharmaceutical and diagnostic companies to develop best practice guidelines for the optimization and validation of mIHC/mIF assays across platforms.
RESULTS
Representative outputs and the advantages and disadvantages of mIHC/mIF approaches, such as multiplexed chromogenic IHC, multiplexed immunohistochemical consecutive staining on single slide, mIF (including multispectral approaches), tissue-based mass spectrometry, and digital spatial profiling are discussed.
CONCLUSIONS
mIHC/mIF technologies are becoming standard tools for biomarker studies and are likely to enter routine clinical practice in the near future. Careful assay optimization and validation will help ensure outputs are robust and comparable across laboratories as well as potentially across mIHC/mIF platforms. Quantitative image analysis of mIHC/mIF output and data management considerations will be addressed in a complementary manuscript from this task force.
Topics: Fluorescent Antibody Technique; Humans; Immunohistochemistry; Immunotherapy; Staining and Labeling; Tumor Microenvironment
PubMed: 32414858
DOI: 10.1136/jitc-2019-000155