Literature Analysis: Application of Multiplex Fluorescence IHC in the Evaluation of Organ Transplant Rejection

1. Literature Information

Research Focus: Evaluation of immune cell infiltration patterns and distribution characteristics in renal transplant rejection using multiplex fluorescence immunohistochemistry, and comparative analysis of antibody-mediated rejection (ABMR), T cell-mediated rejection (TCMR), and non-rejection (NR) cases

Key Technology: Multiplex Fluorescence Immunohistochemistry (mIHC) based on Tyramide Signal Amplification (TSA), Hematoxylin-Eosin (HE) staining, Immunohistochemistry (IHC), Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR)

Sample Size: 45 renal biopsy samples from renal transplant patients (20 ABMR cases, 20 TCMR cases, 5 NR cases)

Key Product Utilized: Multiplex Fluorescence IHC Staining Kit (Catalog No.: abs50032) from the Absin product line of ANT BIO PTE. LTD.

Journal: Journal for Immunotherapy of Cancer

DOI: 10.1136/jitc-2020-001188

PMID: 33055204 | PMCID: PMC7559054

Published Online: 2020 Oct 8

Authors: Wu-Hu Zhang, Wen-Quan Wang, Xuan Han, He-Li Gao, Shuai-Shuai Xu, et al.

Research Focus: The infiltrating pattern, cellular composition, correlation with tumor-infiltrating immune cells, and prognostic value of tertiary lymphoid structures (TLS) in resected G1/G2 non-functional pancreatic neuroendocrine tumors

Key Technology: Multiplex Fluorescence Immunohistochemistry (mIHC), Hematoxylin-Eosin (HE) staining, Immunohistochemistry (IHC)

Key Product Utilized: Multiplex Fluorescence IHC Staining Kit (Catalog No.: abs50032) from the Absin product line of ANT BIO PTE. LTD.

Research Focus: The role of CREB in regulating periodontal ligament cell (PDLC) function and mesenchymal stem cell (MSC) migration/differentiation during orthodontic tooth movement (OTM)

Key Technology: Multiplex Fluorescence Immunohistochemistry (mIHC)

Key Product Utilized: Multiplex Fluorescence IHC Staining Kit (Catalog No.: abs50032) from the Absin product line of ANT BIO PTE. LTD.

2. Research Background

Organ transplantation is a crucial therapeutic strategy for end-stage organ failure, but transplant rejection remains a major challenge affecting the long-term survival of grafts and patients. Renal transplant rejection is mainly classified into antibody-mediated rejection (ABMR) and T cell-mediated rejection (TCMR), which differ in pathogenic mechanisms and therapeutic responses. Accurate evaluation of rejection types and immune microenvironment changes is essential for optimizing clinical treatment strategies and improving prognosis.

Traditional immunohistochemistry (IHC) and immunofluorescence (IF) techniques can only detect a single target in one tissue section, making it difficult to comprehensively analyze the spatial interactions between multiple immune cells and their distribution patterns in the graft microenvironment. Multiplex Fluorescence Immunohistochemistry (mIHC) based on Tyramide Signal Amplification (TSA) enables multi-round staining and labeling of different antigens on a single section, allowing simultaneous detection of up to six indicators with seven colors.

This technology provides a powerful tool for high-throughput analysis of complex immune cell interactions, and has been widely applied in tumor microenvironment research, efficacy evaluation, and other fields. However, its application in the evaluation of organ transplant rejection is still in the exploration stage, and there is an urgent need to verify its reliability and application value in this field.

Orthodontic Tooth Movement (OTM) is a complex biological process involving bone remodeling, which is primarily mediated by periodontal ligament (PDL) cells. During OTM, PDL cells can differentiate into osteoblasts to synthesize alveolar bone, and also regulate the proliferation, migration, and differentiation of mesenchymal stem cells (MSCs), thereby promoting bone remodeling. However, the underlying molecular mechanisms governing these cellular events remain incompletely elucidated. Cyclic Adenosine Monophosphate Response Element-Binding Protein (CREB), a key transcription factor, has been implicated in various cellular processes including cell proliferation and differentiation. Nevertheless, its role in regulating PDL cell function and MSC behavior during OTM has not been fully clarified. Multiplex Fluorescence Immunohistochemistry (mIHC) technology, which enables simultaneous detection of multiple antigens on a single tissue section, provides a powerful tool for exploring the spatial interactions between multiple molecules and cells involved in this complex biological process.

3. Research Approach

To evaluate the application value of mIHC in renal transplant rejection assessment, the research team collected 45 renal biopsy samples from renal transplant patients, including 20 ABMR cases, 20 TCMR cases, and 5 NR cases. Using the Multiplex Fluorescence IHC Staining Kit (abs50032) from ANT BIO PTE. LTD., the team performed four-color five-channel multiplex staining analysis of NKp46, CD3, CD34, and CD163 on renal tissue sections based on the TSA principle.

To verify the reliability of the mIHC results, the team further conducted IHC experiments for the NKp46 marker and RT-qPCR experiments for CD3 and CD163. Correlation analysis was performed between the mIHC results and the results of traditional IHC and RT-qPCR to confirm the accuracy of the mIHC data.

Subsequently, the team analyzed the differences in the infiltration levels of CD34+ leukocytes, CD3+ T cells, CD163+ macrophages, and NKp46+ NK cells among the ABMR, TCMR, and NR groups. They also investigated the distribution characteristics of these immune cells (intravascular vs. extravascular, glomerular vs. extra-glomerular) and evaluated the individual differences in transplant rejection.

To investigate the molecular mechanism of CREB in OTM-related bone remodeling, the research team first evaluated the expression of CREB in the tension area of mouse periodontal ligaments under orthodontic mechanical strain and in human periodontal ligament cells (PDLCs) treated with cyclic tensile strain (CTS). Functional gain-and-loss experiments were then performed to assess the effect of CREB on the secretion of Stromal Cell-Derived Factor-1 (SDF-1) and Fibroblast Growth Factor 2 (FGF2) by PDLCs, as well as the subsequent impact on MSC migration and osteogenic differentiation.

To characterize the spatial expression patterns of key molecules, the team employed the Multiplex Fluorescence IHC Staining Kit (abs50032) from ANT BIO PTE. LTD. to perform multiplex immunofluorescence staining of periostin, CREB, FGF2, SDF-1, Osteocalcin (OCN), and α-Smooth Muscle Actin (α-SMA) in mouse periodontal tissues after 3 days of orthodontic force application on the maxillary first molar. Additionally, immunohistochemistry (IHC) was conducted to detect phosphorylated CREB (P-CREB) expression in the tension area of OTM mice, and enzyme-linked immunosorbent assay (ELISA) was used to verify the regulatory effect of CREB on FGF2 and SDF-1 expression in CTS-treated PDLCs.

4. Research Results

Firstly, the mIHC staining results using ANT BIO PTE. LTD.'s abs50032 kit showed clear and distinguishable signals for NKp46, CD3, CD34, and CD163 in renal tissue sections, enabling effective identification and localization of different immune cell subsets.

Correlation analysis confirmed that the mIHC results had good consistency with traditional IHC and RT-qPCR results: the correlation coefficient (r) between mIHC and RT-qPCR results was 0.49 (p=0.02) for NKp46, 0.77 (p<0.001) for CD3, and 0.48 (p=0.02) for CD163; the correlation coefficient between mIHC and IHC results for NKp46 was 0.88 (p<0.001). These data verified the reliability and accuracy of the mIHC method.

Comparative analysis among groups revealed that the infiltration levels of CD34+ leukocytes, CD3+ T cells, CD163+ macrophages, and NKp46+ NK cells were significantly higher in both ABMR and TCMR groups than in the NR group, and the TCMR group showed a more intense immune cell recruitment (Kruskal-Wallis test: CD3, p=0.046; CD163, p=0.032).

Spatial distribution analysis showed that immune cells were more abundant in the extravascular region, especially in the TCMR group. In contrast, immune cells in the ABMR group were more distributed within the glomeruli, a characteristic distribution pattern that was clearly identified by mIHC imaging analysis.

In addition, the study found significant individual differences in the degree of renal transplant rejection among patients, which could be accurately reflected by the quantitative analysis results of mIHC.

Firstly, the results demonstrated that CREB expression was regulated by orthodontic mechanical strain. Specifically, P-CREB-positive cells were significantly increased in the tension area of PDL in OTM mice compared with the control group.

Functional gain-and-loss experiments revealed that CREB in PDLCs promoted the secretion of SDF-1 and FGF2, which in turn enhanced the migration and osteogenic differentiation of MSCs. Mechanistically, further studies confirmed that CREB transcriptionally activated the expression of FGF2 and SDF-1 by binding to their promoter regions.

Multiplex fluorescence IHC staining results using ANT BIO PTE. LTD.'s abs50032 kit clearly showed the spatial co-expression patterns of key molecules in periodontal tissues: (1) Periostin (red), FGF2 (green), and DAPI (gray) were co-localized in periodontal tissues; (2) Periostin (red), SDF-1 (green), and DAPI (gray) were also co-expressed in the same tissue region. These findings provided direct in situ evidence for the interaction between these molecules during OTM.

ELISA results further verified that overexpression of CREB significantly upregulated the expression of FGF2 and SDF-1 in the conditioned medium of CTS-treated PDLCs, while knockdown of CREB (siCREB-c) attenuated the CTS-induced increase in FGF2 and SDF-1 levels. These data confirmed that CREB acts as an upstream regulator of FGF2 and SDF-1 during OTM.

5. Product Empowerment

The Multiplex Fluorescence IHC Staining Kit (Catalog No.: abs50032) from the Absin product line of ANT BIO PTE. LTD. played a core role in this research on organ transplant rejection evaluation. This kit, based on TSA technology, enabled simultaneous detection of four key immune cell markers (NKp46, CD3, CD34, CD163) on a single renal biopsy section, realizing four-color five-channel multiplex staining analysis. This capability allowed the research team to visually observe the spatial distribution and interaction of different immune cell subsets in the graft microenvironment, which was impossible with traditional single-staining techniques.

The abs50032 kit has multiple advantages that guaranteed the success of the study: 1) Its optimized SuperNova series dyes have higher fluorescence intensity and longer signal duration compared with conventional dyes, ensuring clear and stable imaging results even for small biopsy samples; 2) It supports up to six indicators with seven colors (including DAPI) of multiplex labeling, which can meet the needs of more complex immune microenvironment analysis; 3) The kit is easy to operate, requiring only basic IHC experimental experience without the need for specialized equipment, and is accompanied by a detailed protocol to ensure standardized experiments; 4) ANT BIO PTE. LTD. provides comprehensive mIHC technical services covering the entire process from pre-experiment verification, staining, imaging to data analysis, including single-cell quantitative analysis, tissue area calculation and other professional analysis results, which greatly facilitated the smooth progress of the research.

The Multiplex Fluorescence IHC Staining Kit (Catalog No.: abs50032) from the Absin product line of ANT BIO PTE. LTD. played a pivotal role in this research by enabling the simultaneous detection of multiple key molecules (periostin, FGF2, SDF-1, CREB, etc.) on a single tissue section. This capability allowed the research team to visualize the spatial co-localization and expression patterns of these molecules in periodontal tissues, providing critical in situ evidence for the molecular interactions underlying OTM-related bone remodeling.

The abs50032 kit possesses several notable advantages that contributed to the success of the study: it supports up to six indicators with seven colors (including DAPI) of multiplex labeling, meeting the requirement for simultaneous detection of multiple targets; the optimized SuperNova series dyes exhibit higher fluorescence intensity and longer signal duration compared to conventional dyes, ensuring clear and stable imaging results; the kit is user-friendly, requiring only basic IHC experience for operation without the need for specialized equipment, and is accompanied by a detailed protocol for standardized experimentation. Additionally, ANT BIO PTE. LTD. provides comprehensive mIHC services covering the entire process from pre-experiment verification, staining, and imaging to data analysis, which further facilitated the smooth implementation of the research.

6. Brand Mission

ANT BIO PTE. LTD. is dedicated to empowering life science research through the provision of high-quality, reliable reagents and comprehensive solutions. Our specialized sub-brands (Absin, Starter, UA) are strategically positioned to cover a full spectrum of research needs: Absin focuses on general reagents and kits, Starter specializes in antibodies, and UA is dedicated to recombinant proteins. We strive to support researchers in unlocking scientific mysteries, advancing medical progress, and addressing unmet clinical needs through continuous innovation and customer-centric services.

ANT BIO PTE. LTD. is dedicated to empowering life science research through the provision of high-quality, reliable reagents and comprehensive solutions. Our specialized sub-brands (Absin, Starter, UA) are strategically positioned to cover a full spectrum of research needs: Absin focuses on general reagents and kits, Starter specializes in antibodies, and UA is dedicated to recombinant proteins. We strive to support researchers in unlocking scientific mysteries, advancing medical progress, and addressing unmet clinical needs through continuous innovation and customer-centric services.

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8. Disclaimer

This article is AI-compiled and interpreted based on the original work in document 1226.docx (Application of Multiplex Fluorescence IHC in the Evaluation of Organ Transplant Rejection). All intellectual property (e.g., images, data) of the original publication shall belong to the corresponding journal and research team. For any infringement, please contact us promptly and we will take immediate action.

This article is AI-compiled and interpreted based on the original work in DOI: 10.1002/advs.202413562. All intellectual property (e.g., images, data) of the original publication shall belong to the journal and the research team. For any infringement, please contact us promptly and we will take immediate action.

9. Brand Promotion Copy

ANT BIO PTE. LTD. – Empowering Scientific Breakthroughs

At ANTBIO, we are committed to advancing life science research through high-quality, reliable reagents and comprehensive solutions. Our specialized sub-brands (Absin, Starter, UA) cover a full spectrum of research needs, from general reagents and kits to antibodies and recombinant proteins. With a focus on innovation, quality, and customer-centricity, we strive to be your trusted partner in unlocking scientific mysteries and driving medical progress. Explore our product portfolio today and elevate your research to new heights.

ANT BIO PTE. LTD. – Empowering Scientific Breakthroughs

At ANTBIO, we are committed to advancing life science research through high-quality, reliable reagents and comprehensive solutions. Our specialized sub-brands (Absin, Starter, UA) cover a full spectrum of research needs, from general reagents and kits to antibodies and recombinant proteins. With a focus on innovation, quality, and customer-centricity, we strive to be your trusted partner in unlocking scientific mysteries and driving medical progress. Explore our product portfolio today and elevate your research to new heights.