Journal of Histochemistry & Cytochemistry最新文献

Neutral buffered formalin (NBF) is the most common fixative in clinical applications. However, NBF damages proteins and nucleic acids, limiting the quality of proteomic and nucleic acid-based assays. Prior studies have demonstrated that BE70, a fixative of buffered 70% ethanol, has many benefits over NBF but the degradation of proteins and nucleic acids in archival paraffin blocks remain a challenge. Thus, we evaluated the addition of guanidinium salts to BE70 with the hypothesis that this may protect RNA and protein. Guanidinium salt supplemented BE70 (BE70G)-fixed tissue is comparable with that of BE70 via histology and immunohistochemistry. Western blot analysis also revealed that HSP70, AKT, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression signals in BE70G-fixed tissue were higher than those in BE70-fixed tissue. The quality of nucleic acids extracted from BE70G-fixed, paraffin-embedded tissue was also superior, and BE70G provides improved protein and RNA quality at shorter fixation times than its predecessors. The degradation of proteins, AKT and GAPDH, in archival tissue blocks is also decreased with the addition of guanidinium salt to BE70. In conclusion, BE70G fixative improves the quality of molecular analysis with more rapid fixation of tissue and enhanced long-term storage of paraffin blocks at room temperature for evaluation of protein epitopes.

Pressure ulcers represent a crucial clinical problem, especially in hospitalized patients. Ischemia-reperfusion (I-R) is an important cause of these lesions. Natural killer (NK), invariant NK T (iNKT), and dendritic epidermal T-cells, which express the natural killer group 2, member D (NKG2D) receptor, have been reported to have physiological roles in skin tissue repair and wound healing. However, a role for NKG2D-NKG2D ligand interactions in I-R-induced skin injury has not been determined. Using a murine pressure ulcer model, we demonstrated that I-R-induced ulcers in NKG2D-deficient mice were larger than those in wild-type or T-cell receptor δ knockout mice. Histopathological evaluation revealed that accumulation of macrophages and neutrophils at the peripheral deep dermis and subcutaneous tissue of the ulcers was enhanced in NKG2D-deficient mice. Rae-1 mRNA, which encodes an NKG2D ligand, was induced, and RAE-1 protein was detected immunohistochemically in fibroblasts and inflammatory cells in the dermis after reperfusion. RAE-1 expression was also increased in primary mouse fibroblasts treated with sodium arsenite. These results suggested that NKG2D ligand expression was induced by oxidative stress after I-R injury and support a putative role for this ligand in wound repair. Furthermore, the influx of NKG2D-positive cells at I-R sites may mitigate pressure ulcers via NKG2D-NKG2D ligand interactions.

We tried to prevent nonspecific nuclear staining (NS-NS) of picrosirius red (PSR) staining by treating the specimens with one of the heteropoly acids phosphotungstic acid (PTA). We analyzed a total of 35 cases of non-cancerous liver tissue for fibrosis and NS-NS under PSR-alone, phosphomolybdic acid (PMA)-pretreated PSR (PMA + PSR), or PTA-pretreated PSR (PTA + PSR) condition. In addition, we analyzed the photosensitivity of PMA or PTA single stain specimens. PTA + PSR significantly suppressed NS-NS compared with PSR. The color of the specimens did not change into blue by 30 times the exposure to whole slide scanner (WSS) light. The PTA + PSR condition showed the highest correlation with the Ishak score (pathological evaluation of liver fibrosis) compared with other conditions. Furthermore, Sirius Red-positive percentage (SRP%) in PSR was increased in the NS-NS observed cases. SRP% in PMA + PSR was significantly affected by WSS light exposure time. Moreover, the deposition of non-polarized PSR-stained substances (NP-PSR⁺S) clinging to the collagen fibers potentially explains why SRP% seemed bigger under PSR than PTA + PSR. Our protocol enabled us to analyze the whole slide image of PSR staining by high magnification, which would contribute to the accurate analysis of collagen amount in the tissue sections.

This commentary briefly reviews the background for the development of the horseradish peroxidase-diaminobenzidine tetrahydrochloride histochemical method originally described by Graham and Karnovsky in their citation classic, reprinted in full in this issue of Journal of Histochemistry & Cytochemistry. Some of the method's subsequent applications, including its use as a macromolecular tracer for kidney glomerular permeability and use in immunoelectron microscopy and other immunoassays, are also discussed.

Immunocytochemical (ICC) techniques are frequently used in basic and clinical research. Here, we focus on the importance of using antisera/antibodies at optimal dilutions to achieve specificity and reduce costs. Unfortunately, the basic principle, the necessity to test method specificity of the staining by a series of increasing dilutions of primary antiserum/antibodies, is only occasionally seen in papers using ICC. Many researchers rely on the company's information or others' published data. In this study, we show examples with monoclonal antibodies used in the peroxidase-based ICC technique in mouse and guinea pig brain sections. We show images of ICC staining of phospho-S129 alpha-synuclein in A53T mice and NeuN in guinea pig brains and demonstrate that optimal staining with them can be achieved at least at two to three orders of magnitude higher dilutions than generally used in the literature. We strongly recommend that when antisera/antibodies are used for the first time in any laboratory, independent of what the manufacturer or vendor recommends or are found in the literature, a dilution curve should be set up to identify the optimal dilution. This practice provides not only the highest specificity but is also an economic approach.

Aging leads to substantial structural changes in the skin. Elastic fibers maintain skin structure, but their degeneration and loss of function with age result in wrinkle formation and loss of skin elasticity. Oxytalan fiber, a type of elastic fiber, extends close to the dermal-epidermal junction (DEJ) from the back of the dermis. Oxytalan fibers are abundant in the papillary layer and contribute to skin elasticity and texture. However, to accurately understand the mechanisms of skin elasticity, the interaction between elastic fibers and DEJ should be elucidated. Here, we investigated elastic fibers and DEJ and their structural alterations with aging. Several basement membrane proteins [collagen (COL) IV, COLVII, and laminin 332], fibrous tropoelastin, and fibrillin-1 in excised human skin tissue were observed using three-dimensional imaging. Age-related alterations in COLVII, elastic fibers, and fibrillin-1 were evaluated. We found that COLVII forms long hanging structures and is co-localized with fibrous tropoelastin in young skin but not aged skin. Fibrillin-1-rich regions were observed at the tips of elastin fibers in young skin tissue, but rarely in aged skin. This co-localization of elastic fiber and COLVII may maintain skin structure, thereby preventing wrinkling and sagging. COLVII is a potential therapeutic target for skin wrinkling.

The observation that the two active forms of proprotein convertase 1/3 (PC1/3) were differentially expressed in beta cells of normal islets raised the possibility that this heterogeneity is lost during type 1 diabetes (T1D) progression. To test this hypothesis, the expression of the convertase was evaluated by confocal microscopy in sections of human pancreas of autoantibody positive (AA+) and T1D donors and compared with that of control. Islets of T1D pancreas were comprised of beta cells expressing either low or high PC1/3 levels and all islets of a pancreatic section contained only one beta cell type. Pancreata of AA+ donors contained either of these two classes of islets intermixed with normal islets comprised of beta cells with heterogeneous PC1/3 expression. This alteration affected the expression of proinsulin and insulin, which in most AA+ and T1D donors were lower than in controls. The present results indicate that the heterogeneity of PC1/3 expression is lost in all beta cells in a subset islets of AA+ donors and in all islets of T1D donors. These findings suggest that the heterogeneity of PC1/3 expression is a biomarker of human beta cell health and that its loss coincides with the initial stages of T1D.