Fig 1: Flow cytometric detection, isolation, and characterization of autofluorescent events in adult cow and human ovarian cortical tissue. (A–D) Representative gating strategy for doublet discrimination (forward-scatter or FSC-A: B; side-scatter or SSC-A: C) and for dead cell exclusion using 4',6-diamidino-2-phenylindole (DAPI) labeling (D). (E, F) Comparison of autofluorescent events detected in the APC-A far-red channel (640-nm laser; E) versus the PE-Texas red-A channel (561-nm laser; F). (G–K): Autofluorescent events detected in the PE-Texas red-A channel were collected, fixed and permeabilized (G and H), and then incubated with APC-conjugated primary antibodies against SMA (Abcam ab5694) or CD31 (Invitrogen MA3100) (I and J) for determination of the total percentage of autofluorescent events that were positive for expression of either PVC marker in bovine and human ovarian cortical tissue samples (K). Data shown in (K) are the mean ±SE; n = 4 (CD31) or 7 (SMA), and n = 5 (SMA) or 6 (CD31), for bovine and human sample analysis, respectively.
Fig 2: Right ventricular systolic pressure measured by right heart catheterization (a), RV/(LV + septum) by gross weight (b), the percentage of remodeled vessels in a lung section (c), and immunohistochemical analysis of a-smooth muscle actin (a-SMA) staining in the smooth muscle layer of small pulmonary arteries of the lungs ((d) to (g)). For right heart pressure analysis, the catheter was inserted into the jugular vein and guided into the right ventricle to measure right ventricular systolic pressure (RVSP). Animals were culled, the heart was isolated, atria were removed and RV/(LV + septum) were measured to assess RV hypertrophy. Although there are significant differences between the normoxic group (n = 8) and Sugen–hypoxic groups at five (n = 4) and eight weeks (n = 4), there were no significant differences in RVSP nor RV/(LV + septum) at autopsy between Sugen–hypoxia five and eight weeks. Vascular thickening was determined by smooth muscle actin antibody (ab5694, Abcam, Cambridge, UK) staining, thickening was characterized by an increase in the vessel wall diameter of more than 50% of the arterial wall or complete occlusion. The number of remodeled vessels over the total number of vessels present in a lung section was determined. Results are shown as mean ± SEM. ANOVA test was used to compare the three groups, and if there was statistical significance, a Tukey HSD test was used for post hoc analysis. * represents p < 0.05 and ** represents p < 0.01. For immunohistochemical analysis of a-smooth muscle actin (a-SMA) staining in the smooth muscle layer of small pulmonary arteries of the lung, sections were viewed at ×200. (d) and (f) demonstrate normoxic animals at five and eight weeks while (e) and (g) demonstrate vascular thickening and remodeling of the pulmonary vasculature (black arrows) in Sugen–hypoxic rats at five and eight weeks, respectively.RVSP: RV systolic pressure; SuHx: Sugen–hypoxia; RV: right ventricle; LV: left ventricle.
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