Abstract. This document is the consensus of the American Association of Veterinary Laboratory Diagnosticians (AAVLD) Subcommittee on Standardization of Immunohistochemistry on a set of guidelines for immunohistochemistry (IHC) testing in veterinary laboratories. Immunohistochemistry is a powerful ancillary methodology frequently used in many veterinary laboratories for both diagnostic and research purposes. However, neither standardization nor validation of IHC tests has been completely achieved in veterinary medicine. This document addresses both issues. Topics covered include antibody selection, fixation, antigen retrieval, antibody incubation, antibody dilutions, tissue and reagent controls, buffers, and detection systems. The validation of an IHC test is addressed for both infectious diseases and neoplastic processes. In addition, storage and handling of IHC reagents, interpretation, quality control and assurance, and troubleshooting are also discussed. Proper standardization and validation of IHC will improve the quality of diagnostics in veterinary laboratories.

Objective-To compare the chondrogenic potential of adult equine mesenchymal stem cells derived from bone marrow (MSCs) or adipose tissue (ASCs).Study Design-In vitro experimental study. Animals-Adult Thoroughbred horses (n = 11).Methods-BM (5 horses; mean [± SD] age, 4 ± 1.4 years) or adipose tissue (6 horses; mean age, 3.5 ± 1.1 years) samples were obtained. Cryopreserved MSCs and ASCs were used for pellet cultures in stromal medium (C) or induced into chondrogenesis ± transforming growth factor-3 (TGFβ 3 ) and bone morphogenic factor-6 (BMP-6). Pellets harvested after 3, 7, 14, and 21 days were examined for cross-sectional size and tissue composition (hematoxylin and eosin), glycosaminoglycan (GAG) staining (Alcian blue), collagen type II immunohistochemistry, and by transmission electron microscopy. Pellet GAG and total DNA content were measured using dimethylmethylene blue and Hoechst DNA assays.Results-Collagen type II synthesis was predominantly observed in MSC pellets from Day 7 onward. Unlike ASC cultures, MSC pellets had hyaline-like matrix by Day 14. GAG deposition occurred earlier in MSC cultures compared with ASC cultures and growth factors enhanced both MSC GAG concentrations (P<.0001) and MSC pellet size (P<.004) after 2 weeks in culture. Conclusion-EquineMSCs have superior chondrogenic potential compared with ASCs and the equine ASC growth factor response suggests possible differences compared with other species.Clinical Relevance-Elucidation of equine ASC and MSC receptor profiles will enhance the use of these cells in regenerative cartilage repair.

BackgroundFetal stress has been linked to adult atherosclerosis, obesity, and diabetes. Epidemiology studies have associated fetal exposure to maternal smoking and postnatal exposure to environmental tobacco smoke (ETS) with increased asthma risk.ObjectiveWe tested the hypothesis, in a mouse model of asthma, that in utero ETS exposure alters airway function and respiratory immune responses in adults.MethodsPregnant Balb/c mice were exposed daily to ETS or HEPA-filtered air (AIR). Offspring inhaled aerosolized ovalbumin (OVA) or saline in weeks 7–8. Regardless of whether they inhaled OVA or saline, mice were sensitized by OVA injections in weeks 11 and 13 followed by OVA aerosol challenge in weeks 14–15. At three time points, we assessed OVA-specific serum immunoglobins, bronchoalveolar lavage cells and cytokines, lung and nasal histopathology, and airway hyperresponsiveness (AHR).ResultsAt 6 weeks, we found no significant differences between in utero ETS and AIR mice. At 10 weeks, following OVA aerosol, ETS mice displayed greater AHR than AIR mice (α = 0.05), unaccompanied by changes in histopathology, cytokine profile, or antibody levels. At 15 weeks, mice that had inhaled saline in weeks 7–8 developed airway inflammation: eosinophilia (α = 0.05), interleukin-5 (α = 0.05), and AHR (α = 0.05) were greater in ETS mice than in AIR mice. Mice that had inhaled OVA in weeks 7–8 demonstrated no airway inflammation after sensitization and challenge.ConclusionIn utero ETS exposure exacerbates subsequent adult responses to initial allergen exposure.