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Percent of positively stained cells was estimated based on the fractional area of staining within the total cluster area.
Remaining GUS stain was analyzed for intensity and area of staining using the 'volume analysis' tool from BioRad Gel-Doc imaging software.
Compared to uninjected embryos (Figure 6C) or hjv morphants (Figure 6D), mtp2 morphants exhibited increased staining intensity for hepcidin in the foregut, but a smaller area of staining in the liver (Figure 6E).
Each case was scored on the basis of the intensity and area of staining.
Standard procedures were used for visualisation and the staining was quantified using a semiquantitative scale (1 4) that evaluated both the intensity and area of staining.
Under light microscopy (Olympus), the distribution of phospho-LYRIC in the nucleus and cytoplasm of the cells was rated according to a score that determined the scale of intensity of staining to the area of staining using ImageMaster software.
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The volume fraction of collagen was determined by measuring the area of stained LV within a given field.
Interstitial collagen volume fraction was determined by measuring the area of stained tissue within a given field, excluding vessels, artefacts, minor scars or incomplete tissue fields; 15 20 fields were analysed per animal in a blinded manner.
Areas of staining with no discrete breaks counted as a single vessel.
The lateral and medial femoral condyles show both degeneration of the articular surface and areas of staining loss relative to the surrounding tissue.
The whole section was examined to determine area and intensity of staining.
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