After fixation, whole-mounted mammary glands were processed through an alcohol series, defatted with toluene, stained with carmine alum, dehydrated in an alcohol and xylene series, and preserved in k-pax heat-sealed bags (Thermo Fisher Scientific) with methyl salicylate (Acros Organics, Morris Plains, NJ) [51 (link)]. Two digital images of whole-mount mammary glands (one from the left; one from the right third pectoral glands) were obtained using an AxioImager dissection microscope (Carl Zeiss Microscopy, Jena, Germany) at ×30 magnification and a Zeiss high-resolution color camera. To evaluate mammary gland morphology, ZEN software (Carl Zeiss Microscopy) was used to measure ductal density [51 (link)]. A 13 × 16 grid (180 crosshairs, 0.3 mm apart) was placed on each image, and the fraction of crosshairs that fell on ducts was quantified and averaged for the two images for each sample.
Protocol detail
Quantification of Mammary Gland Ductal Density
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Alum
Carmine
Dehydrated alcohol
Dissection
Mammary gland
Methyl salicylate
Microscopy
Toluene
Xylene
Corresponding Organization : University of Massachusetts Amherst
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Variable analysis
independent variables
- None explicitly mentioned
- Ductal density
- Alcohol series
- Toluene defatting
- Carmine alum staining
- Alcohol and xylene series dehydration
- Preservation in k-pax heat-sealed bags with methyl salicylate
- Positive control: Not mentioned
- Negative control: Not mentioned
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