Review

Authors: Kyuseok Im, Sergey Mareninov, M Fernando Palma Diaz...

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

Review

Authors: Wei Chang Colin Tan, Sanjna Nilesh Nerurkar, Hai Yun Cai...

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

Review

Authors: Wenjie Sheng, Chaoyu Zhang, T M Mohiuddin...

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

Review

Authors: Benedetta Terziroli Beretta-Piccoli, Giorgina Mieli-Vergani, Diego Vergani...

Circulating autoantibodies are a key diagnostic tool in autoimmune hepatitis (AIH), being positive in 95% of the cases if tested according to dedicated guidelines issued by the International Autoimmune Hepatitis Group. They also allow the distinction between type 1 AIH, characterized by positive anti-nuclear and/or anti-smooth muscle antibody, and type 2 AIH, characterized by positive anti-liver kidney microsomal type 1 and/or anti-liver cytosol type 1 antibody. Anti-soluble liver antigen is the only AIH-specific autoantibody, and is found in 20-30% of both type 1 and type 2 AIH. Anti-neutrophil cytoplasmic antibody is frequently positive in type 1 AIH, being associated also with inflammatory bowel disease and with primary/autoimmune sclerosing cholangitis. The reference method for autoantibody testing remains indirect immunofluorescence on triple tissue (rodent liver, kidney and stomach), allowing both the detection of the majority of liver-relevant reactivities, including those autoantibodies whose molecular target antigens are unknown. Of note, the current knowledge of the clinical significance of autoantibodies relies on studies based on this technique. However, immunofluorescence requires trained laboratory personnel, is observer-dependent, and lacks standardization, leading to ongoing attempts at replacing this method with automated assays, the sensitivity, and specificity of which, however, require further studies before they can be used as a reliable alternative to immunofluorescence; currently, they may be used as complementary to immunofluorescence.

Topics: Antibodies, Antineutrophil Cytoplasmic; Autoantibodies; Fluorescent Antibody Technique, Indirect; Hepatitis, Autoimmune; Humans; Immunologic Tests

PubMed: 34491531
DOI: 10.1007/s12016-021-08888-9

Authors: Ian D Odell, Deborah Cook

Topics: Antigen-Antibody Complex; Autoantibodies; Fluorescent Antibody Technique; Humans; Skin Diseases

PubMed: 23299451
DOI: 10.1038/jid.2012.455

Authors: Amélie Viratham Pulsawatdi, Stephanie G Craig, Victoria Bingham...

Multiplex immunofluorescence is a powerful tool for the simultaneous detection of tissue-based biomarkers, revolutionising traditional immunohistochemistry. The Opal methodology allows up to eight biomarkers to be measured concomitantly without cross-reactivity, permitting identification of different cell populations within the tumour microenvironment. In this study, we aimed to validate a multiplex immunofluorescence workflow in two complementary multiplex panels and evaluate the tumour immune microenvironment in colorectal cancer (CRC) formalin-fixed paraffin-embedded tissue. We stained CRC and tonsil samples using Opal multiplex immunofluorescence on a Leica BOND RX immunostainer. We then acquired images on an Akoya Vectra Polaris and performed multispectral unmixing using inform. Antibody panels were validated on tissue microarray sections containing cores from six normal tissue types, using qupath for image analysis. Comparisons between chromogenic immunohistochemistry and multiplex immunofluorescence on consecutive sections from the same tissue microarray showed significant correlation (r  > 0.9, P-value < 0.0001), validating both panels. We identified many factors that influenced the quality of the acquired fluorescent images, including biomarker co-expression, staining order, Opal-antibody pairing, sample thickness, multispectral unmixing and biomarker detection order during image analysis. Overall, we report the optimisation and validation of a multiplex immunofluorescence process, from staining to image analysis, ensuring assay robustness. Our multiplex immunofluorescence protocols permit the accurate detection of multiple immune markers in various tissue types, using a workflow that enables rapid processing of samples, above and beyond previous workflows.

Topics: Epitopes; Fluorescent Antibody Technique; Humans; Imaging, Three-Dimensional; Reproducibility of Results; Tumor Microenvironment; Workflow

PubMed: 32671911
DOI: 10.1002/1878-0261.12764

Review

Authors: Seema Chhabra, Ranjana Walker Minz, Biman Saikia...

Direct immunofluorescence (DIF) and indirect immunofluorescence (IIF) tests on skin biopsy are being done mostly in academic teaching hospitals. These tests provide a useful diagnostic aid to dermatologists. Immunohistology and serology can, in conjunction with histology, provide considerable help in delineation and diagnosis of various skin disorders as well as systemic diseases with skin involvement, e.g. systemic lupus erythematosus. Immunofluorescence (IF) studies have now become an invaluable supplement to clinical and histological examination in a variety of dermatological diseases. These skin diseases now include not only bullous and connective tissue disorders, vasculitides, and conditions such as lichen planus, but also the scaling dermatoses, notably psoriasis. In this review article, we share our experience of providing such a diagnostic facility for more than 30 years in a large tertiary care health center in North India and also help to outline the conditions, which can be diagnosed confidently, and others where IF can help in confirming a diagnosis or the immune component of the disease. The article also deals with handling of skin biopsy specimens and interpretation of biopsy findings on DIF and IIF examination.

Topics: Biopsy; Fluorescent Antibody Technique, Direct; Fluorescent Antibody Technique, Indirect; Humans; Skin; Skin Diseases; Specimen Handling

PubMed: 23075636
DOI: 10.4103/0378-6323.102355

Authors: Sigrid Fra-Bido, Simon A Walker, Silvia Innocentin...

Location of immune cells that form the germinal center reaction within secondary lymphoid tissues can be characterized using confocal microscopy. Here, we present an optimized immunofluorescence staining protocol to image germinal center structures in fixed/frozen spleen sections from ChAdOx1 nCoV-19 immunized mice. This protocol can be adapted to identify other cell types within secondary lymphoid tissues. For complete information on the generation and use of this protocol to examine immune responses to the COVID vaccine ChAdOx1 nCoV-19, please refer to Silva-Cayetano et al. (2020).

Topics: Animals; COVID-19; ChAdOx1 nCoV-19; Fluorescent Antibody Technique; Germinal Center; Immunization, Secondary; Immunogenicity, Vaccine; Male; Mice; SARS-CoV-2; Spleen

PubMed: 34195671
DOI: 10.1016/j.xpro.2021.100499

Review

Authors: Wonjin Cho, Sehun Kim, Young-Gyun Park...

Profiling molecular expression in situ allows the integration of biomolecular and cellular features, enabling an in-depth understanding of biological systems. Multiplexed immunofluorescence methods can visualize tens to hundreds of proteins from individual tissue samples, but their application is usually limited to thin tissue sections. Multiplexed immunofluorescence of thick tissues or intact organs will enable high-throughput profiling of cellular protein expression within 3D tissue architectures (e.g., blood vessels, neural projections, tumors), opening a new dimension in diverse biological research and medical applications. We will review current multiplexed immunofluorescence methods and discuss possible approaches and challenges to achieve 3D multiplexed immunofluorescence.

Topics: Proteins; Fluorescent Antibody Technique

PubMed: 37131224
DOI: 10.1186/s13041-023-01027-9

Review

Authors: Hazem A Juratli, Pinar Avci, Barbara Horváth...

Pemphigus comprises a heterogeneous group of autoimmune blistering diseases, which can affect both skin and mucous membranes, especially oral mucosa. This group of diseases shows usually a chronic-relapsing course. Since pemphigus is a rare disease, the diagnosis is often delayed, because it is based upon the recognition of consistent clinical, histologic, and direct immunofluorescence findings, as well as indirect immunofluorescence, and/or enzyme-linked immunosorbent assay. Usually the patients are treated for multiple other conditions before starting the correct therapy, leading to a critical reduction of the patients' quality of life. This review is a succinct compilation of pearls gathered from clinical experience in pemphigus and the myths that may have influenced everyday practice but have been proven false. This review provided a selection of such dilemmas and controversies, focusing on myths and pearls that can help young dermatologist in the clinic, while also dispelling them.

Topics: Autoimmune Diseases; Blister; Fluorescent Antibody Technique, Indirect; Humans; Pemphigus; Quality of Life

PubMed: 33960749
DOI: 10.23736/S2784-8671.21.06810-3