Immunohistochemical Markers for Prospective Studies in Neurofibromatosis-1 Porcine Models

Abstract

Neurofibromatosis type 1 (NF1) is a common, cancer-predisposing disease caused by mutations in the NF1 tumor gene. Patients with NF1 have an increased risk for benign and malignant tumors of the nervous system (e.g., neurofibromas, malignant peripheral nerve sheath tumors, gliomas) and other tissues (e.g., leukemias, rhabdomyosarcoma, etc.) as well as increased susceptibility to learning disabilities, chronic pain/migraines, hypertension, pigmentary changes, and developmental lesions (e.g., tibial pseudoarthrosis). Pigs are an attractive and upcoming animal model for future NF1 studies, but a potential limitation to porcine model research has been the lack of validated reagents for direct translational study to humans. To address that issue, we used formalin-fixed tissues (human and pigs) to evaluate select immunohistochemical markers (activated caspase-3, allograft inflammatory factor-1, beta-tubulin III, calbindin D, CD13, CD20, desmin, epithelial membrane antigen, glial fibrillary acidic protein, glucose transporter-1, laminin, myelin basic protein, myoglobin, proliferating cell nuclear antigen, S100, vimentin, and von Willebrand factor). The markers were validated by comparing known expression and localization in human and pig tissues. Validation of these markers on fixed tissues will facilitate prospective immunohistochemical studies of NF1 pigs, as well as other pig models, in a more efficient, reproducible, and translationally relevant manner.

Allograft Inflammatory Factor 1 as an Immunohistochemical Marker for Macrophages in Multiple Tissues and Laboratory Animal Species

“…Immunohistochemistry for AIF1 (previously known as ionized calcium-binding adapter molecule 1, IBA1; or IFNγ-responsive transcript 1) was performed as previously validated for the detection of microglia in the brains of humans and pigs. 30 Briefly, formalin-fixed, paraffin-embedded tissues were sectioned (thickness, approximately 4 µm), placed on glass slides (Superfrost Plus, Fisher Scientific, Pittsburgh, PA), baked (60 °C, 60 min; Isotemp Oven, Fisher Scientific), and hydrated through a series of xylene and alcohol baths. Heatinduced epitope retrieval was performed in a citrate buffer bath at pH 6.0 (125 °C, 5 min; model DC2002, Decloaking Chamber, BioCare Medical, Concord, CA).…”

“…However, optimized and validated immunohistochemical techniques that are applicable across species might be advantageous not only for potential significance and translation but also for cost efficiency in the laboratory. 11,30,36 Inflammation is a common and critical component of many diseases 7,31 and is morphologically characterized by the presence of immune cells at histopathology. Macrophages are important immune cells that are quiescent residents in tissues and become activated through different pathways to mediate host defense and inflammation.…”

Longitudinal phenotype development in a minipig model of neurofibromatosis type 1

“…Tumor tissues were fixed (10% neutral buffered formalin x 5-7 days), processed, paraffin-embedded, sectioned (~4 µm) and stained for hematoxylin and eosin (HE) or Masson's Trichrome (MT) stains. S100 immunohistochemistry was performed as detailed previously 64 . Tissues were examined by a veterinary pathologist following principles of reproducible histopathological examination and scoring 65 .…”

Validating indicators of CNS disorders in a swine model of neurological disease

“…The following markers (GFAP, IBA1, and MBP) have been validated in the cerebrum of wildtype swine previously [23]. However, these antibodies have not been validated in other areas of the brain that are commonly affected in neurological diseases.…”