Genotypically de-identified images were analyzed using ImageJ (Abramoff et al., 2004 ) for DNase I, C4 and AC15 for specific stages of oogenesis. Follicle staging was assigned based on morphology and size.
DNase I nucleolar to cytoplasmic ratios were quantified from single confocal slices in S7/8 follicles by measuring the integrated density of fluorescence within a square in the nucleolus, compared to a square in the adjacent cytoplasm; the focal planes chosen had the strongest nucleolar DNase I signal. Three paired measurements were made per cell and the average nucleolar/cytoplasmic ratio was determined. Three cells per follicle were measured. DNase I data were analyzed and statistical analysis performed using Prism (Graphpad, RRID:SCR_002798 ).
Quantification of C4 nucleolar actin was performed on confocal image stacks of follicles stained with anti-actin C4, WGA, and Phalloidin; as necessary, brightness and contrast were adjusted to score all the C4 nucleolar actin present. Data were collected for S5-6, S7-8, and S9 follicles. For each follicle the number of nurse cells exhibiting structured nucleolar C4 actin was scored.
Quantification of AC15 nuclear actin level and nucleolar puncta presence/size was performed on confocal image stacks of follicles stained with anti-actin AC15, WGA, and DAPI. For AC15 nuclear actin level, data were collected for S5-6, S7-8, and S9 follicles. For each follicle the nurse cells and the follicle cells were scored for their level of AC15 staining on a 5-point scale ranging from background levels typical of what is observed in wild-type S3 follicles (-) to the strongest staining typical of what is observed in wild-type S10 follicles (+++). For AC15 puncta, data were collected for S7/8, S9, and S10 follicles; as necessary, brightness, contrast and zoom were adjusted to score the puncta. Each follicle was scored as having either no AC15 puncta, small puncta, or large/obvious puncta in the nurse cells. C4 and AC15 data were analyzed using Excel (Microsoft, RRID:SCR_016137 ) and statistical analysis was preformed using R (Vienna, Austria, RRID: 001905).
DNase I nucleolar to cytoplasmic ratios were quantified from single confocal slices in S7/8 follicles by measuring the integrated density of fluorescence within a square in the nucleolus, compared to a square in the adjacent cytoplasm; the focal planes chosen had the strongest nucleolar DNase I signal. Three paired measurements were made per cell and the average nucleolar/cytoplasmic ratio was determined. Three cells per follicle were measured. DNase I data were analyzed and statistical analysis performed using Prism (Graphpad, RRID:
Quantification of C4 nucleolar actin was performed on confocal image stacks of follicles stained with anti-actin C4, WGA, and Phalloidin; as necessary, brightness and contrast were adjusted to score all the C4 nucleolar actin present. Data were collected for S5-6, S7-8, and S9 follicles. For each follicle the number of nurse cells exhibiting structured nucleolar C4 actin was scored.
Quantification of AC15 nuclear actin level and nucleolar puncta presence/size was performed on confocal image stacks of follicles stained with anti-actin AC15, WGA, and DAPI. For AC15 nuclear actin level, data were collected for S5-6, S7-8, and S9 follicles. For each follicle the nurse cells and the follicle cells were scored for their level of AC15 staining on a 5-point scale ranging from background levels typical of what is observed in wild-type S3 follicles (-) to the strongest staining typical of what is observed in wild-type S10 follicles (+++). For AC15 puncta, data were collected for S7/8, S9, and S10 follicles; as necessary, brightness, contrast and zoom were adjusted to score the puncta. Each follicle was scored as having either no AC15 puncta, small puncta, or large/obvious puncta in the nurse cells. C4 and AC15 data were analyzed using Excel (Microsoft, RRID:
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