Mechanically isolated follicles cultured for 24 or 72 hours in the presence or
absence of 10 nM DHT were formalin fixed and set in 2% (weight/volume)
low-melting-point agarose (Sigma) before being dehydrated through an alcohol series,
embedded in wax, and serially sectioned (5 µm) onto Superfrost charged slides
(VWR, Lutterworth, United Kingdom). Immunofluoresence detection of AR was carried out
as described later, and one central section of each follicle was selected for
analysis. Confocal images of individual follicles were imported into ImageJ and split
into red-green-blue channels. Blue 4′,6-diamidino-2-phenylindole
(DAPI)–labeled nuclei were thresholded (same threshold value for all
follicles) to produce a binary image (white DAPI, positive; black DAPI, negative) and
define the nuclei. Nuclei were outlined using the “Analyze Particles”
command. In the green channel, AR labeling was thresholded (with the same threshold
maintained for all follicles), the nuclear outlines were overlaid, and the area of
labeling exceeding the threshold was measured within the nuclear outlines, and for
the GC layer as a whole. The proportion of nuclear area or GC area that exceeded the
threshold (i.e., the proportion of the nuclei, or GC layer, that was
AR positive) was compared for control and DHT-treated follicles at 24 and 72
hours.
absence of 10 nM DHT were formalin fixed and set in 2% (weight/volume)
low-melting-point agarose (Sigma) before being dehydrated through an alcohol series,
embedded in wax, and serially sectioned (5 µm) onto Superfrost charged slides
(VWR, Lutterworth, United Kingdom). Immunofluoresence detection of AR was carried out
as described later, and one central section of each follicle was selected for
analysis. Confocal images of individual follicles were imported into ImageJ and split
into red-green-blue channels. Blue 4′,6-diamidino-2-phenylindole
(DAPI)–labeled nuclei were thresholded (same threshold value for all
follicles) to produce a binary image (white DAPI, positive; black DAPI, negative) and
define the nuclei. Nuclei were outlined using the “Analyze Particles”
command. In the green channel, AR labeling was thresholded (with the same threshold
maintained for all follicles), the nuclear outlines were overlaid, and the area of
labeling exceeding the threshold was measured within the nuclear outlines, and for
the GC layer as a whole. The proportion of nuclear area or GC area that exceeded the
threshold (i.e., the proportion of the nuclei, or GC layer, that was
AR positive) was compared for control and DHT-treated follicles at 24 and 72
hours.