Abstract
Immunohistochemistry (IHC) has become an essential component of diagnostic and experimental pathology. But with all such tools, it is critical to understand the limitations so that results are not erroneously interpreted. One of the limiting factors of IHC is thought to be prolonged fixation in 10% neutral-buffered formalin. Prolonged formalin fixation can result in epitope masking, with a subsequent decrease in immunoreactivity. Although antigen retrieval techniques can increase antigen detection, a systematic study to evaluate the time-dependent effects of formalin fixation on IHC has not been performed for most antibodies currently used in domestic animals. In this report the authors evaluated the effects of various time periods of formalin fixation on immunohistochemical results using 61 different antigen-antibody combinations. Tissues submitted to a veterinary diagnostic lab were fixed in formalin and removed after 1 day, 3 days, or at 1-week intervals up to 7 weeks. The vast majority of tissues were from canines. IHC was performed with a wide assortment of antibodies, including various cytokeratins, CD markers commonly used to immunophenotype hematologic neoplasms, Glut-1, laminin, somatostatin, and many others. Antigen retrieval methods were used in most IHC protocols. Most antibodies had moderate to strong immuostaining at all time points examined, up to 7 weeks of formalin fixation. However, cytokeratin 7, laminin, and high-molecular-weight cytokeratins had markedly reduced immunoreactivity after 3 days, 2 weeks, and 6 weeks of formalin fixation, respectively. The authors conclude that in general, formalin fixation up to 7 weeks is not a major concern for most IHC protocols used in veterinary diagnostic pathology. Exceptions to this rule, however, necessitate careful evaluation of the effects of formalin fixation time during antibody validation procedures.
Webster JD, Miller MA, DuSold D, Ramos-Vara J.
Amyloid fibrils arranged in β-pleated sheets are associated with a variety of pathological conditions such as amyloidosis, diabetes and Alzheimer's disease, but these structures also have normal functions in some mammalian organisms. The authors of this paper hypothesized that peptide and protein hormones in neuroendocrine cells are stored in secretory granules in an amyloid-like aggregation. They randomly tested 42 peptide and protein hormones from different organs and different species and discovered that 10 of these spontaneously formed amyloid fibrils in vitro. However, when the same peptides and proteins were incubated with low molecular weight heparin (to represent glycosaminoglycans), 31 formed amyloid fibrils. Two hormones from the same prohoromone had different propensities to form amyloid fibrils. Adrenocorticotropic hormone (ACTH) did not form amyloid, while β-endorphin did. The authors then determined that the amyloid formation of β-endorphin is necessary for aggregation of ACTH in secretory granules. They conclude that functional amyloid in the pituitary and other organs likely plays a role in normal cellular physiology and this should be considered when evaluating abnormal amyloid formation. Their work also sheds light on normal processes of storage and release of hormones from granules in neuorendocrine tissues.
Maji SK, Perrin MH, Sawaya MR.
The Balb/c mouse strain is generally considered to be resistant to development of spontaneous vasculitis. During the course of experiments involving femoral artery ligation, the authors detected arteritis at the base of the aorta with incidence ranging from 18–56% of mice. Vasculitis was not present in any other organs examined. In order to determine if this was a heretofore unreported background lesion in this strain or if it was secondary to ischemia from the femoral artery manipulation, they repeated the femoral ligation experiments on 20 mice and added 20 control mice. Aortitis was present in both control (n = 3) and treated (n = 2) mice, with a cumulative incidence of 12.5%. The microscopic changes were identical in both groups and consisted of a focal to circumferential, transmural, mixed infiltrate with macrophages, PMNs, and lymphocytes. Thrombi were occasionally present, but none of the mice had fibrinoid vascular necrosis or vasculitis in any other organs. Operated mice had higher blood levels of IL-6 than controls, but this was not correlated with the presence of vasculitis. The presence of spontaneous aortitis in Balb/c mice must be considered when evaluating the cardiovascular system in future experiments.
Ramot Y, Manno RA, Okazaki Y, et al.
The laryngeal muscles of animals differ in the subsets of myosin heavy chains that can be expressed. Thus far, the myosin heavy chains and muscle fiber types present in normal and diseased equine laryngeal muscles have not been well characterized. In this paper, the authors performed an extensive immunohistochemical analysis of laryngeal muscles from normal horses and those with subclinical recurrent laryngeal neuropathy (RLN). They found that the cricoarytenoideus dorsalis and arytenoideus transverses muscles of horses without evidence of RLN have the same myosin heavy chain profile as seen in normal equine limbs, with slow, 2a and 2× fibers present. Three of the horses had fiber-type grouping in the left cricoarytenoideus dorsalis muscle, which earns them the ‘subclinical’ RLN designation. In these horses there was an absence of 2× fibers and a marked increase in 2a and slow fibers, along with hypertrophy of muscles fibers. Together, these results suggest that early equine RLN may be due to a combination of pathophysiological mechanisms, including selective denervation and reinnervation of type 2× fibers with conversion to type 2a or slow fibers, disruption of the neural impulses that regulate the various muscle fibers, and compensatory hypertrophy the muscle fibers that remain. The compensatory hypertrophy may allow the horses to persist in the subclinical phase until the compensatory mechanism is exhausted.
Rhee HS, Steele CM, Derksen FJ, et al.