All animal experiments were performed in accordance with The Guide for the Care and Use of Laboratory Animals: Eighth Edition (National Research Council, National Academy Press, Washington, DC, USA, 2011) and under the protocol (NO. 108658) approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Toledo, Toledo, OH. Three-week-old female BALB/c mice (n = 18) were purchased from the Jackson Laboratory (Bar Harbor, MN) and housed under a 12-h light-dark cycle with ad libitum access to regular chow and water. Starting at the age of 4 weeks old, mice received intraperitoneal injection of 100 µL of a drug (vehicle: PBS, L-arginine (20 mg/kg) or L-NAME (20 mg/kg)) every other day for 6 weeks. At the end of the treatment period, mice were euthanized, and inguinal (number 5) mammary glands were harvested and processed for paraffin-embedding and sectioning. Most paraffin sections were deparaffinized, hydrated and analyzed by immunohistochemistry. Other sections were analyzed by SHG imaging to visualize stromal collagen density (see below).

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