Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Protocol for investigating tertiary lymphoid structures in human and murine fixed tissue sections using Opal™-TSA multiplex immunohistochemistry.

ABSTRACT: Tertiary lymphoid structures (TLSs) are ectopic lymphoid aggregates that can develop within or adjacent to tumors, but protocols that can accurately identify and characterize TLSs are lacking. Here, we present a protocol for the in situ interrogation and characterization of TLSs in human and murine tissue sections using Opal™-tyramide signal amplification multiplex immunohistochemistry. This protocol enables simultaneous detection of up to 7 markers (6 antigens and a DAPI counterstain). We also describe a grading system to identify immature and mature TLSs.

SUBMITTER: Quigley LT

PROVIDER: S-EPMC9843255 | biostudies-literature | 2023 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Protocol for investigating tertiary lymphoid structures in human and murine fixed tissue sections using Opal™-TSA multiplex immunohistochemistry.

Quigley Luke T LT Pang Lokman L Tavancheh Elnaz E Ernst Matthias M Behren Andreas A Huynh Jennifer J Da Gama Duarte Jessica J

STAR protocols 20230110 1

Tertiary lymphoid structures (TLSs) are ectopic lymphoid aggregates that can develop within or adjacent to tumors, but protocols that can accurately identify and characterize TLSs are lacking. Here, we present a protocol for the in situ interrogation and characterization of TLSs in human and murine tissue sections using Opal™-tyramide signal amplification multiplex immunohistochemistry. This protocol enables simultaneous detection of up to 7 markers (6 antigens and a DAPI counterstain). We also ...[more]

PMID: 36633948

Similar Datasets

Improvement of Opal Multiplex Immunofluorescence Workflow for Human Tissue Sections.

Project description:The Opal multiplex technique is an established methodology for the detection of multiple biomarkers in one section. The protocol encompasses iterative single stainings and heating-mediated removal of the primary and secondary antibodies after each staining round, leaving untouched the Opal fluorophores which are deposited onto the antigen of interest. According to our experience, repetitive heating of skin sections often results in tissue damage, indicating an urgent need for milder alternatives to strip immunoglobulins. In this study, we demonstrate that considerable heating-related damage was found not only in skin but also in tissues of different origin, mostly characterized by low cell density. Importantly, the morphology remained fully intact when sections were repetitively exposed to β-mercaptoethanol-containing stripping buffer instead of multiple heating cycles. However, target epitopes appeared sensitive at a differential degree to multiple treatments with stripping buffer, as shown by loss in staining intensity, but in all cases, the staining intensity could be restored by increment of the primary antibody concentrations. Application of β-mercaptoethanol-containing stripping buffer instead of heating for antibody removal markedly improved the quality of the Opal multiplex technique, as a substantial higher number of differently colored cells could be visualized within a well-conserved morphological context.

| S-EPMC8091416 | biostudies-literature

Cyclic Multiplex Fluorescent Immunohistochemistry Protocol to Phenotype Glial Cells in Formalin-Fixed Paraffin-Embedded Human Brain Sections.

Project description:There is a growing interest in expanding the multiplexing capability of immunohistochemistry to achieve a deeper phenotyping of various cell types in health and disease. Here, we describe a protocol of cyclic multiplex fluorescent immunohistochemistry that enables the labeling of up to 16 antigens on the same formalin-fixed paraffin-embedded section using "off-the-shelf," commercially available, primary antibodies as well as fluorescently conjugated secondary antibodies. Key steps include the denaturing/stripping of the antibodies by microwaving and the quenching of any remaining fluorescent signal between the cycles of otherwise traditional multiplexed fluorescent immunohistochemistry. We have successfully applied this protocol to characterize astrocytic and microglial responses to Aβ plaques and neurofibrillary tangles in Alzheimer's disease brains, but it could be easily adapted to other user's needs regarding cell types, disease, and organ.

| S-EPMC9900949 | biostudies-literature

Multiplex quantitative measurement of mRNAs from fixed tissue microarray sections.

Project description:The development of prognostic and diagnostic biomarkers, such as those from gene expression studies, requires independent validation in clinical specimens. Immunohistochemical analysis on tissue microarrays (TMAs) of formalin-fixed paraffin-embedded tissue is often used to increase the statistical power, and it is used more often than in situ hybridization, which can be technically limiting. Herein, we introduce a method for performing quantitative gene expression analysis across a TMA using an adaptation of 2D-RT-qPCR, a recently developed technology for measuring transcript levels in a histologic section while maintaining 2-dimensional positional information of the tissue sample. As a demonstration of utility, a TMA with tumor and normal human prostate samples was used to validate expression profiles from previous array-based gene discovery studies of prostate cancer. The results show that 2D-RT-qPCR expands the utility of TMAs to include sensitive and accurate gene expression measurements.

| S-EPMC4022353 | biostudies-literature

Improved Multiplex Immunohistochemistry for Immune Microenvironment Evaluation of Mouse Formalin-Fixed, Paraffin-Embedded Tissues.

Project description:Immune profiling of tissue through multiplex immunohistochemistry is important for the investigation of immune cell dynamics, and it can contribute to disease prognosis and evaluation of treatment response in cancer patients. However, protocols for mouse formalin-fixed, paraffin-embedded tissue have been less successful. Given that formalin fixation and paraffin embedding remains the most common preparation method for processing mouse tissue, this has limited the options to study the immune system and the impact of novel therapeutics in preclinical models. In an attempt to address this, we developed an improved immunohistochemistry protocol with a more effective Ag-retrieval buffer. We also validated 22 Abs specific for mouse immune cell markers to distinguish B cells, T cells, NK cells, macrophages, dendritic cells, and neutrophils. In addition, we designed and tested novel strategies to identify immune cells for which unique Abs are currently not available. Last, in the 4T1 model of breast cancer, we demonstrate the utility of our protocol and Ab panels in the quantitation and spatial distribution of immune cells.

| S-EPMC6310091 | biostudies-literature

Investigating mammary glands of lactating goats for the presence of tertiary lymphoid organs.

Project description:Ectopic tertiary lymphoid organs (TLOs) have been identified in many organs, such as the lungs, nasal cavities, and kidneys of both mice and humans. Although lymphocyte aggregates have been observed in the mammary glands of ruminants, the details remain unclear. In this study, we investigated the mammary glands of lactating goats for the presence of TLOs. The localization of CD20 (B cells), CD3 (T cells), MECA79 (high endothelial venules), CD40 (follicular dendritic cells), BCL6 (germinal center), and IgA was examined by immunohistochemistry. The concentrations of IgG, IgA, lactoferrin, β-defensin-1, cathelicidin-2, cathelicidin-7, S100A7, and S100A8 in milk were measured by ELISA. The localization and amount of tight junction (TJ) proteins (claudin-3 and claudin-4) were examined using immunofluorescence and western blotting. We found that 19 out of 30 udders contained lymphocyte aggregates, which showed positive reactions against CD20, CD3, CD40, and MECA79. In addition, large-sized aggregations showed separate localization of B cells and T cells and a positive reaction against BCL6, although BCL6 was sparsely localized in the aggregations. These results indicate that mammary glands of lactating goats contain TLOs. The IgG and IgA concentrations in the milk of TLO-positive goats and the number of IgA-positive cells were higher than those in negative goats. Furthermore, claudin-4 was localized in the TJ region and the amount was higher in TLO-positive mammary glands than that in the negative group, indicating the presence of leakages at TJs. In conclusion, a majority of lactating goat udders have TLOs, which contribute to local immunity by producing immunoglobulins.

| S-EPMC9399771 | biostudies-literature

Global Protein Profiling in Processed Immunohistochemistry Tissue Sections.

Project description:Tissue sections, which are widely used in research and diagnostic laboratories and have already been examined by immunohistochemistry (IHC), may subsequently provide a resource for proteomic studies, even though only small amount of protein is available. Therefore, we established a workflow for tandem mass spectrometry-based protein profiling of IHC specimens and characterized defined brain area sections. We investigated the CA1 region of the hippocampus dissected from brain slices of adult C57BL/6J mice. The workflow contains detailed information on sample preparation from brain slices, including removal of antibodies and cover matrices, dissection of region(s) of interest, protein extraction and digestion, mass spectrometry measurement, and data analysis. The Gene Ontology (GO) knowledge base was used for further annotation. Literature searches and Gene Ontology annotation of the detected proteins verify the applicability of this method for global protein profiling using formalin-fixed and embedded material and previously used IHC slides.

| S-EPMC10379013 | biostudies-literature

Protocol to characterize mouse intestinal epithelial cell lineage using Opal multiplex immunofluorescence.

Project description:Applying Opal multiplex immunofluorescence (OMI) to characterize intestinal tissues of genetically engineered mouse models provides an excellent tool for studying complex processes. However, detecting appropriate signals from multiple target molecules is challenging. Here, we present a protocol to characterize mouse intestinal epithelial cell lineage using OMI. We describe steps for processing small intestine and colonic mouse tissues and designing and optimizing panels for OMI in mouse intestinal tissues. We then detail procedures for performing a quantitative evaluation of acquired images. For complete details on the use and execution of this protocol, please refer to Kinoshita et al.1.

| S-EPMC11422768 | biostudies-literature

API2-MALT1 fusion transcripts involved in mucosa-associated lymphoid tissue lymphoma: multiplex RT-PCR detection using formalin-fixed paraffin-embedded specimens.

Project description:Malignant lymphoma of mucosa-associated lymphoid tissue (MALT) type is a distinct clinicopathological disease entity in the category of extranodal marginal zone B-cell lymphoma. Recently, we and others have shown that the API2 gene on chromosome 11 and the MALT1 gene on chromosome 18 are fused as a result of t(11;18)(q21;q21) in MALT lymphomas. Here we report a detection assay that can be used for formalin-fixed, paraffin-embedded specimens. It consists of a multiplex one-tube reverse transcriptase-polymerase chain reaction (RT-PCR) followed by three parallel multiplex nested polymerase chain reactions. Eight variants of the fusion transcripts have been reported to date. When these variants were used as positive controls, all were successfully detected. The subsequent direct sequencing confirmed the results. Using this rapid and simple method, we could detect API2-MALT1 fusion transcripts in 5 of 15 (33%) archival cases of MALT lymphoma for a frequency comparable with those of RT-PCR assays using frozen materials. The lung was the preferential anatomical site of origin of MALT lymphomas harboring API2-MALT1 fusion. No fusion transcript was detected in any of 20 high-grade B-cell lymphomas. Our multiplex RT-PCR assay, which can be used for routinely-processed paraffin samples, should serve as a useful molecular tool for clarifying the clinicopathological significance of API2-MALT1 fusion in MALT lymphoma.

| S-EPMC1850327 | biostudies-literature

Multiplex immunohistochemistry accurately defines the immune context of metastatic melanoma.

Project description:A prospective study explored the heterogeneous nature of metastatic melanoma using Multiplex immunohistochemistry (IHC) and flow cytometry (FACS). Multiplex IHC data quantitated immune subset number present intra-tumoral (IT) vs the tumor stroma, plus distance of immune subsets from the tumor margin (TM). In addition, mIHC showed a close association between the presence of IT CD8+ T cells and PDL1 expression in melanoma, which was more prevalent on macrophages than on melanoma cells. In contrast, FACS provided more detailed information regarding the T cell subset differentiation, their activation status and expression of immune checkpoint molecules. Interestingly, mIHC detected significantly higher Treg numbers than FACS and showed preferential CD4+ T cell distribution in the tumor stroma. Based on the mIHC and FACS data, we provide a model which defines metastatic melanoma immune context into four categories using the presence or absence of PDL1+ melanoma cells and/or macrophages, and their location within the tumor or on the periphery, combined with the presence or absence of IT CD8+ T cells. This model interprets melanoma immune context as a spectrum of tumor escape from immune control, and provides a snapshot upon which interpretation of checkpoint blockade inhibitor (CBI) therapy responses can be built.

| S-EPMC6057961 | biostudies-literature

Increased Angiogenesis and Lymphangiogenesis in Adenomyosis Visualized by Multiplex Immunohistochemistry.

Project description:There is evidence for increased angiogenesis in the (ectopic) endometrium of adenomyosis patients under the influence of vascular endothelial growth factor (VEGF). VEGF stimulates both angiogenesis and lymph-angiogenesis. However, information on lymph vessels in the (ectopic) endometrium of adenomyosis patients is lacking. In this retrospective matched case-control study, multiplex immunohistochemistry was performed on thirty-eight paraffin embedded specimens from premenopausal women who had undergone a hysterectomy at the Amsterdam UMC between 2001 and 2018 to investigate the evidence for (lymph) angiogenesis in the (ectopic) endometrium or myometrium of patients with adenomyosis versus controls with unrelated pathologies. Baseline characteristics of both groups were comparable. In the proliferative phase, the blood and lymph vessel densities were, respectively, higher in the ectopic and eutopic endometrium of patients with adenomyosis than in the endometrium of controls. The relative number of blood vessels without α-smooth muscle actinin (α SMA) was higher in the eutopic and ectopic endometrium of adenomyosis patients versus controls. The level of VEGF staining intensity was highest in the myometrium but did not differ between patients with adenomyosis or controls. The results indicate increased angiogenesis and lymphangiogenesis in the (ectopic) endometrium affected by adenomyosis. The clinical relevance of our findings should be confirmed in prospective clinical studies.

| S-EPMC9369277 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data