Cdkn1b Knockout Impacts Mammary Gland Development

Mammary glands from female Cdkn1b^+/+ and Cdkn1b^-/- groups (both N1xN1 and N6xN6) were harvested and whole mounts were generated as described previously [30 (link)]. The inguinal and abdominal mammary glands were collected, fixed, stained in carmine, dehydrated and cleared in xylene. Samples were imaged on a Nikon Ti/E inverted microscope using Nikon Elements software. Histology and multicolor immunofluorescence analyses were performed as described previously [3 (link)]. After heat-induced antigen retrieval in TRIS-EDTA buffer (pH 9), the samples were blocked with 5% goat serum PBS and stained with antibodies against Ki67 (BD550609, 1:50), Milk-specific proteins (Nordic-MUbio, RAM/MSP, 1:200), PR (ab16661, 1:100), FoxA1 (ab23738, 1:200) and phospho-Stat5 (AbCam ab194898, 1:250). Images from the stained sections were obtained by Nikon inverted widefield live-cell imaging system. The percentage of cells for each marker was estimated by counting positive cells and total cells per field from 4 to 6 randomly selected regions using ImageJ 1.45s software. Luminal ectasia was scored by measuring the percentage area of milk protein to mammary epithelium.

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Ding L., Shunkwiler L.B., Harper N.W., Zhao Y., Hinohara K., Huh S.J., Ekram M.B., Guz J., Kern M.J., Awgulewitsch A., Shull J.D., Smits B.M, & Polyak K. (2019). Deletion of Cdkn1b in ACI rats leads to increased proliferation and pregnancy-associated changes in the mammary gland due to perturbed systemic endocrine environment. PLoS Genetics, 15(3), e1008002.

Publication 2019

Ti/E inverted microscope ab16661 ab194898

Abdominal Antibodies Antigen Carmine Cdkn1b Cells Ectasia Edta Epithelium Female Foxa1 Goat Immunofluorescence Inguinal Mammary Microscope Milk protein Serum Stat5 Tris buffer Xylene

Corresponding Organization : Medical University of South Carolina

Other organizations : University of Wisconsin–Madison

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Variable analysis

independent variables

Genotype: Cdkn1b+/+ and Cdkn1b-/- groups

dependent variables

Mammary gland morphology and development (measured through whole mount analysis)
Proliferation (measured by Ki67 staining)
Differentiation (measured by milk-specific protein staining)
Progesterone receptor (PR) expression
FoxA1 expression
Phospho-Stat5 expression
Luminal ectasia (measured by the percentage area of milk protein to mammary epithelium)

control variables

Mouse strains: N1xN1 and N6xN6 backgrounds

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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