Granulysin was also expressed in a large percentage of CD8-positive cells ( Fig. While granulysin also localized to CD56-positive cells in AUB endometrial tissue, the percentage was higher (85% ± 2.8%) however, this difference was not statistically significant ( P>.05) between groups. As depicted in Figure 5, the granulysin protein (green) colocalized with that of CD56 (uNK cells, red) in approximately 68% ± 4.1% (mean ± SEM) of the CD56-positive cells of control patients ( Fig. 5, left panel). Because granulysin is a major cytolytic and proinflammatory molecule produced by both CD56-positive (uNK) and CD8-positive (CTLs) cells, we performed colocalization/dual immunohistochemical localization for granulysin and these uNK/CTL markers in endometrial tissue from both controls and AUB subjects. In accord with the elevated granulysin protein expression, granulysin mRNA expression was significantly higher in endometrial biopsy tissue from patients with AUB ( Fig. 4). To determine if the increased granulysin protein localization/expression was associated with the increased granulysin mRNA expression, we assessed the transcript levels in FFPE sections from the same specimens evaluated for granulysin protein expression in Figure 3. In accord with the increased expression of uNK cells and CTLs, granulysin expression was significantly greater in endometrial biopsies from patients with AUB (19% vs. Because both cell types express granulysin, we quantitated its expression in endometrial biopsies from the same patient population. 17.2%, Fig. 1) as was the number of CD8-positive cells (presumed to be CTLS) (30.4% vs. The number of CD56-positive cells (presumed to be uNK cells) was significantly greater in endometrial biopsy tissue from women with AUB than in that from controls (40.7% vs. For all IHC, negative controls included the omission of the primary antibody and use of an isotype-matched control ( To determine the expression of GNLY within CD56- and CD8-positive cells, IF IHC was used as described earlier. These 2 numbers were then averaged for each protein in each slide to generate the overall mean percent positive cells per specimen. Two observers (C.I., W.B.N.) each calculated the mean % of positive cells per slide for each protein. For GNLY and CD56, nuclei were identified by hematoxylin staining, whereas for CD8, nuclei were identified by DAPI staining. The number of cells/field expressing each of the 3 proteins of interest were separately calculated as a percentage of total cells/field (number of cells positive for either GNLY, CD56, or CD8 divided by the number of cells expressing nuclear stain). The percent positive cells expressing GNLY, CD56, or CD8 were calculated by assessing signal in 3 to 5 random sections per slide in a 10× high-power field. The signal intensity was strong and consistent for all proteins among all tissue specimens. For all immunohistochemical assessment, localization and the level of signal (protein) intensity were quantitated using the following approach. After coverslipping, slides were stored in the dark at 4☌. To identify nuclei, slides were incubated with 4′,6-diamidino-2-phenylindole (DAPI D3571 Thermo Fisher Scientific) for 5 minutes at 22☌ followed by mounting of slides. The primary antibody was added concurrently to the slides at a 1:250 dilution with the secondary antibodies (goat, anti-rabbit, Alexa Fluor 488 for granulysin goat, anti-rat, Alexa Fluor 647 for CD8 and goat, anti-mouse, Alexa Fluor 546 for CD56). ), the basic procedure was performed as described earlier with the following modifications.
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