Name: अनुभागीय मानव एंडोमेट्रियल ऊतक के ऊतक इम्यूनोहिस्टोकेमिकल धुंधला हो जाना
Uploaded: 2023-10-13T13:50:00
Description: Watch this Scientific Journal Video about Tissue Immunohistochemical Staining of Sectioned Human Endometrial Tissue at JoVE.com

tissue immunohistochemical staining of sectioned human endometrial tissue

Image Analysis of Immunohistochemically Stained Endometrial Tissue

Recurrent miscarriage (RM) is the loss of two or more consecutive pregnancies and is a complex disease drawing attention from clinicians in recent years.The incidence rate of RM in women of childbearing age is 1%-5% 1. Results of previous studies show that immune factors are closely associated with the pathogenesis of RM2,3,4,5. Maintaining immune homeostasis at the maternal-fetal interface is required for embryo implantation and development. Endometrial immune cells perform several regulatory roles to maintain this homeostasis, such as promoting trophoblast invasion, remodeling spiral arteries, and contributing to placenta development6,7,8,9.
Aberrant endometrial immune cells in women with RM have previously been reported. Results show a close association between the high density of uterine natural killer cells (uNKs) and the occurrence of RM10,11,12. An increased number of macrophages has been reported in the endometrium of women with RM, compared with those who had a live birth13. Regulatory T cells (Treg) play a role in maternal immune tolerance toward the embryo, and their level and function are decreased in the decidua of RM patients14. Cytotoxicity T cells (CTL) and dendritic cells (DCs) also play a role in the immune regulation of pregnancy15,16. Therefore, a comprehensive quantitative analysis of local endometrial immune cells during the mid-luteal phase could help to better understand the pathogenesis of RM. Some current methods for quantitative analysis of endometrial immune cells use flow cytometry which can accurately label immune cells with multiple markers17,18. However, clinical application of flow cytometry is limited because it can only be performed on fresh tissue. Obtaining fresh tissue is only feasible when a large volume of excess tumor is available, a rare occurrence for endometrium. Immunohistochemistry can observe tissue morphology well in situ and can also label various immune cells, while traditional immunohistochemical techniques cannot perform quantitative analysis of immune cells.
Compared to conventional immunohistochemistry experiments, quantitative immunohistochemical analysis of immune cells in the endometrium has important clinical significance. IHC intensity scoring is usually ranked on a four-point scale or strong and weak in pathological diagnostics and research19,20,21. However, this semi-quantitative technique is subjective, highly inaccurate, and demonstrates significant intra-observer and inter-observer variability22. One possible solution is the application of machine learning, which is valuable indigital image analysis23,24. By providing quantitative measurements, this approach enables a more precise assessment of immune cell infiltration, distribution, and density within the uterine tissue. This quantitative information can help to elucidate the dynamic changes in immune cell populations during the menstrual cycle and in various pathological conditions. Overall, the ability to quantitatively analyze immune cells in the endometrium through immunohistochemistry offers valuable insights into the immune microenvironment of the uterus.
Therefore, the protocol aimed to developed and validated a digital immunohistochemistry image analysis platform to quantitatively analyze endometrial immune cells including uNK cells, Tregs, macrophages, DCs and cytotoxic T cells during the mid-luteal phase in RM patients.

Author Spotlight: Advancing Reproductive Immunology with a Protocol for the Quantitative Evaluation of Endometrial Immune Cells 

इम्यूनोहिस्टोकेमिस्ट्री और डिजिटल छवि विश्लेषण के आधार पर एंडोमेट्रियल प्रतिरक्षा कोशिकाओं की मात्रात्मक पहचान के लिए मंच

Currently, it is challenging to stabilize the analysis within a certain range. It is also difficult to maintain the quality control of the experiment. Our research can help the patients with abnormal endometrial sensitivity in immune to find the cause of reproductive failure.The local proportion of immune cells in the endometrial can be stably and repeatedly detected. Our findings may promote the research progress of the endometrial sensitivity evaluation in patients with repeated implantation failure.

Watch this Scientific Journal Video about Tissue Immunohistochemical Staining of Sectioned Human Endometrial Tissue at JoVE.com

अनुभागीय मानव एंडोमेट्रियल ऊतक के ऊतक इम्यूनोहिस्टोकेमिकल धुंधला हो जाना

शुरू करने के लिए, पतला एंटीबॉडी समाधान को एक विशेष अभिकर्मक बोतल में स्थानांतरित करें। एक स्वचालित इम्यूनोहिस्टोकेमिकल स्टैंडिंग इंस्ट्रूमेंट के अभिकर्मक डिब्बे में डिटेक्शन किट डालें। अगला, एक स्लाइड धारक पर खंडित एंडोमेट्रियल ऊतक के साथ स्लाइड सेट करें और उन्हें एक विशेष कवर टाइल के साथ कवर करें।फिर धारक को उपकरण के प्रयोगात्मक प्रतिक्रिया डिब्बे में डालें। एक बार जब उपकरण स्वचालित रूप से अभिकर्मक और स्लाइड पर जानकारी को पहचान लेता है, तो इम्यूनोहिस्टोकेमिकल धुंधला शुरू करने के लिए स्टार्ट बटन दबाएं। धुंधला होने के बाद, स्लाइड धारक को बाहर निकालें।साफ पानी के साथ किसी भी शेष डाई को धोने के लिए स्लाइड धारक में सना हुआ स्लाइड रखो। अब, स्लाइड धारक को एक स्वचालित कवर स्लिपर पर रखें और निर्जलीकरण और सीलिंग प्रक्रिया चलाएं। एक बार निर्जलीकरण और सीलिंग पूरा हो गया है स्लाइड निकालें.

Research

JoVE Journal

Biology

Tissue Immunohistochemical Staining of Sectioned Human Endometrial Tissue

Platform for Quantitative Detection of Endometrial Immune Cells Based on Immunohistochemistry and Digital Image Analysis

To evaluate the endometrial immune microenvironment of patients with recurrent miscarriage (RM), a digital immunohistochemistry image analysis platform ...