Immunohistochemical double staining (dual immunohistochemistry, Double IHC) is an advanced pathological detection technique based on conventional single immunohistochemical staining. Its core principle is to specifically label and localize two different target antigens simultaneously on the same tissue section (paraffin section/frozen section). By using two contrasting chromogenic systems to distinguish the signals, it can visually display the expression locations, expression levels, and co-localization relationships of the two proteins. The main difference from the fluorescence method is that image merging is not required; the difference in the colors of the two proteins can be observed directly with the naked eye, and the results can be permanently preserved without worrying about signal degradation.

The multiplex fluorescent immunostaining system, based on sequential staining and antibody elution techniques, combined with automated digital image analysis, overcomes the technical bottlenecks of traditional multiplex immunohistochemistry. It enables high-precision, multi-target immune microenvironment analysis of archived paraffin-embedded tissues, providing novel technical support for studying the immune mechanisms of liver diseases such as hepatic inflammation, liver fibrosis, and hepatocellular carcinoma, while also offering a general experimental protocol for immune phenotypic analysis of multi-organ tissues.

According to the World Health Organization (WHO), herpes simplex virus (HSV) is a prevalent virus, affecting nearly two-thirds of the world's population. Once inside the body, HSV forms a latent infection that periodically awakens, causing painful herpes simplex, usually around the nose and mouth or reproductive area. While HSV is just an annoyance to most people, for some it can also cause dangerous eye infections and brain inflammation, as well as life-threatening infections in newborns.

Herpesviridae refers to a viral family consisting of enveloped double-stranded DNA viruses with large, sophisticated genomes, generally called herpesviruses. These pathogens replicate inside the cell nucleus of numerous vertebrate species. Among over a hundred identified herpesvirus strains, nine distinct types commonly infect human bodies, namely herpes simplex virus 1, herpes simplex virus 2, varicella-zoster virus, Epstein-Barr virus, cytomegalovirus, human herpesvirus-6, human herpesvirus-7, and Kaposi's sarcoma-associated herpesvirus.

Rabies virus (RABV) belongs to the Lyssavirus genus under the Rhabdoviridae family, a neurotropic pathogen that triggers fatal encephalitis across all warm-blooded animal species. Globally, domestic dogs act as the primary natural reservoir and transmission vector of RABV. Human infection mainly occurs when virus-laden saliva from infected animals enters the body via bites or scratches. Timely administration of rabies vaccine combined with human rabies immunoglobulin after exposure is critical to block disease onset, yet there are no targeted antiviral medications capable of directly neutralizing rabies virus particles.

Respiratory Syncytial Virus (RSV) is a non-segmented single-stranded negative-sense RNA pathogen classified under the Pneumovirus genus of the Paramyxoviridae viral family. Its name derives from its unique capacity to fuse adjacent infected host cells together.

The AMPK signaling cascade maintains stable energy metabolism and efficient lipid breakdown across tissues subjected to calorie restriction (CR). Functioning as a universal cellular fuel sensor and metabolic regulator, this signaling axis boosts ATP-generating catabolic pathways while suppressing energy-consuming anabolic reactions in nearly all tissue types.

The cell division cycle (shortened as cell cycle) refers to the full recurring process of eukaryotic proliferative cells from the completion of one cell division to the start of the next. During this whole process, cellular chromosomal DNA undergoes complete accurate duplication, followed by even partitioning of replicated genetic material into two independent daughter cells with identical genetic compositions.

The Forkhead box (Fox) protein superfamily consists of diverse transcription factors unified by a conserved winged-helix DNA-binding structural domain, which enables specific recognition and binding to target gene promoter sequences within cell nuclei.

Glucagon is a key pancreatic peptide hormone that maintains blood glucose homeostasis by triggering hepatic glycogenolysis and gluconeogenesis via the glucagon signaling cascade. This pathway relies on GCGR-coupled Gs/Gq proteins to activate two core downstream branches: the cAMP-PKA axis and calcium-dependent ERK-CREB signaling. Together, these cascades upregulate rate-limiting enzymes such as PEPCK and G6Pase to boost endogenous glucose synthesis and suppress glycogen formation. Abnormal transduction of glucagon signals contributes to hyperglycemia, type 2 diabetes, glucagonoma and obesity-related glucose metabolic disorders. Clarifying the molecular mechanism of this pathway offers valuable theoretical basis for exploring therapeutic targets of glycometabolic diseases.