5.1 Basic Principles of Immunohistochemistry
Immunohistochemistry (IHC) is a critical technique in biomedical research and clinical diagnostics, leveraging the specificity of antibodies to detect particular antigens in tissue sections. The fundamental principle of IHC involves the binding of an antibody to its target antigen, followed by visualization through various detection systems, which can be chromogenic or fluorescent. This process typically begins with the fixation of tissue samples to preserve cellular structures, followed by embedding in paraffin or freezing for cryosectioning. The antibodies used in IHC can be monoclonal or polyclonal, each with unique advantages depending on the specificity and sensitivity required for the study. The use of secondary antibodies conjugated to enzymes or fluorophores enhances the detection signal, allowing for the visualization of antigen distribution and expression levels within the tissue microenvironment. Recent advancements have introduced multiplexed IHC techniques, enabling the simultaneous detection of multiple antigens in a single tissue section, which is particularly beneficial for understanding complex biological processes and cellular interactions in situ [32, 33]. Furthermore, the integration of imaging technologies with IHC has opened new avenues for tissue analysis, providing high-dimensional data that can elucidate the spatial organization and heterogeneity of cellular populations within tissues [34, 35].
5.2 Applications of Immunohistochemistry in KOA Research
In the context of KOA, immunohistochemistry has emerged as a powerful tool for elucidating the pathophysiological mechanisms underlying this degenerative joint disease. IHC allows researchers to visualize and quantify the expression of various biomarkers associated with inflammation, cartilage degradation, and bone remodeling in affected tissues. For example, studies have utilized IHC to investigate the localization and expression levels of pro-inflammatory cytokines, MMPs, and other mediators involved in the OA process [36, 37]. By employing IHC techniques, researchers have been able to identify specific cellular populations, such as macrophages and T-cells, that contribute to the inflammatory milieu in OA-affected joints [38]. Additionally, the application of multiplex IHC in OA research has facilitated the simultaneous assessment of multiple markers, providing insights into the interplay between different cellular pathways and their contributions to disease progression [39, 40]. This comprehensive approach not only enhances the understanding of OA pathogenesis but also aids in the identification of potential therapeutic targets and biomarkers for disease monitoring and progression [41, 42]. Overall, the use of immunohistochemistry in KOA research underscores its significance in advancing our knowledge of this complex condition and improving clinical outcomes for patients.
5.3 The immunohistochemical expression of COX-2 in rat KOA
To determine the immunohistochemical expression of COX-2 in KOA, 30 male SD rats aged 8.5 weeks were selected from the Animal Experiment Center of Guangxi Medical University (License No: SYXK Gui 2020-0004, Ethical Approval No: 202105004). The experimental group (10 rats) underwent no surgical intervention. Twenty SD male rats were anesthetized with 10% chloral hydrate (3 ml/kg) via intraperitoneal injection, and once satisfactory anesthesia was achieved, the rats were placed in a supine position and fixed to the surgical table, followed by routine disinfection and draping. The skin of the left and right hind limbs was shaved with a surgical razor, and the hind limbs were cleaned with gauze soaked in new chlorhexidine, followed by disinfection with iodine. In the model group (10 rats), a 2 cm incision was made parallel to the medial collateral ligament at the left and right knees, with the skin, muscle, and fascia separated in sequence, the joint capsule incised, and the patella displaced at a 90° flexion to open the joint cavity. The anterior cruciate ligament was located and cut with scissors, and a drawer test was performed to ensure complete transection of the anterior cruciate ligament. The joint cavity was flushed with 0.9% sodium chloride solution, and the joint capsule and skin were sutured. In the sham group (10 rats), only the joint capsule was incised without any further treatment, and then sutured. After all rats recovered, they were returned to their cages. One month later, the articular cartilage tissues from each group of rats were dewaxed and activated for enzyme assays, followed by routine paraffin embedding and sectioning.
Knee joint immunohistochemical staining: The paraffin sections were dewaxed to water, followed by antigen retrieval, and placed in a 3% hydrogen peroxide solution for 25 minutes at room temperature in the dark. The slides were washed 3–5 times in PBS (pH 7.4) on a decolorizing shaker, with each wash lasting 5 minutes, and then blocked with serum for 30 minutes. After removing the blocking solution, the prepared primary antibody was added to the sections, which were then incubated in a humid box at 4°C for 12 hours. The corresponding secondary antibody was applied at room temperature for 50 minutes. The slides were washed 3 times in PBS, with each wash lasting 5 minutes. DAB chromogenic solution was added, and the chromogenic time was controlled under a microscope; a brown-yellow color indicated a positive result, after which the slides were rinsed with running water to stop the reaction. The cell nuclei were stained with hematoxylin, dehydrated, and mounted. The expression of COX-2 related antibodies was detected.
In this study, in the control group, COX-2 antibody showed positive expression in immunohistochemical staining.See Fig. 1.
See Fig. 1 Immunohistochemical manifestations of osteoarthritis pathology in SD rats.