Multiplex immunostaining (mIHC) allows the simultaneous detection of multiple antigenic targets within the same tissue section, providing a deeper understanding of spatial variation in cellular distribution. The aim of the present study is to apply this technique to examine the spatial variation of lymphocyte populations in swine lymph nodes during PCV2-SD and PRRSV lymphadenopathy compared with reactive lymphoid hyperplasia. A triple immunohistochemical stain with CD3, CD20 and IBA1 antibodies for the concurrent detection of T lymphocytes, B lymphocytes and macrophages, respectively, was performed. Multiplex immunohistochemistry (mIHC) revealed that, compared to reactive hyperplasia, the most significant changes in lymph node cell populations occurred in the follicles for both PCV2 and PRRSV infections. Additionally, in PCV2 cases, notable alterations were also observed in the interfollicular areas. In PCV2-affected lymph nodes, follicles not only significantly decreased in number but also showed a marked significant reduction in CD20⁺ and CD3⁺ cells. The interfollicular region in these cases also exhibited a significant reduction in CD3⁺ cells. In contrast, PRRSV-associated lymphadenopathy showed significantly increased CD20⁺ cells in the follicles, with a similar trend noted in the interfollicular region. mIHC provides more informative results on a single tissue section, thus preserving the topographical information of the tissue and allowing a comprehensive study of cellular composition, cellular functionality and cell–cell interactions, proving to be a valuable tool for studying and understanding disease dynamics.

Background: Dry Eye Disease (DED) is a multifactorial disorder characterized by tear film instability and ocular surface inflammation. Murine models based on environmental stress are widely used to mimic evaporative DED, although many focus on limited disease features. This study aimed to provide an integrated characterization of ocular surface alterations induced by chronic desiccating stress. Methods: Adult mice were housed in a Controlled-Environmental Chamber (CEC) with low humidity and increased airflow for up to 21 days and sacrificed after 14 or 21 days. Corneal damage was assessed by fluorescein staining. Conjunctival histology was evaluated for epithelial morphology, goblet cell (GC) size, and mucin composition. Complement fractions C3 and C5a were assessed by immunohistochemistry. Expression of inflammatory markers (Major Histocompatibility Complex, Class II, DR, HLA-DR; interleukin-1β, IL-1β; tumor necrosis factor-α, TNF-α) was quantified by Real-Time PCR (RT-PCR) in corneal and conjunctival epithelium. Results: Fluorescein staining revealed progressive corneal epithelial damage over time. Histological analysis demonstrated conjunctival epithelial alterations characterized by a significant reduction in GC size and in neutral mucin-positive GCs, consistent with mucin remodeling of the ocular surface epithelium. Increased epithelial deposition of complement fractions C3 and C5a was observed, while molecular analysis confirmed upregulation of inflammatory markers, including HLA-DR, IL-1β, and TNF-α. Collectively, these findings indicate that the model captures key pathophysiological components of DED. Conclusions: The CEC model reproduces major features of evaporative DED, including epithelial damage, GC remodeling, immune activation, and inflammation. As a non-invasive desiccating stress model, it represents a relevant experimental platform for studying ocular surface inflammation and for preclinical evaluation of therapeutic strategies.

This study aimed to standardize the generation of primary three-dimensional (3D) cell cultures derived from spontaneous canine tumors and to compare two culture systems: a ready-to-use bioreactor (VITVO®) and a miniaturized plate-based system (MPBS). A total of 27 tumors, including mainly hemangiosarcomas, oral melanomas, urothelial carcinomas, and osteosarcomas, were collected from three Italian veterinary hospitals. Fresh samples were used to establish 3D cell cultures and to perform histological and immunohistochemical analyses for phenotypic characterization and comparison with primary tumors. Tissue dissociation proved to be a critical step. A manual protocol combining mechanical and enzymatic methods significantly improved cell viability and cellularity compared to automated dissociation, which yielded fewer viable single cells and limited growth. Both culture systems successfully supported 3D cell cultures using the same scaffold material. The plate-based system was particularly suitable for small biopsy samples without compromising cellularity, whereas VITVO® was more appropriate for larger tissue specimens. An inverse correlation was observed between the extent of necrosis in primary tumors and the cellularity of the corresponding 3D cell cultures. Immunohistochemical analyses confirmed that most 3D cell cultures retained the phenotypic characteristics of the original tumors, while osteosarcoma models exhibited limited growth, likely due to stromal predominance. Overall, these findings highlight the importance of optimizing dissociation protocols, scaffold size, and culture conditions to preserve tumor heterogeneity and viability. This workflow provides a standardized and reproducible platform for generating representative 3D models for comparative oncology research.