Permanent mounting media

After deparaffinization and rehydration, sections were heated to 95 °C for 15 min in Citrate Buffer, pH 6.0 (Invitrogen), for antigen retrieval. Hydrogen peroxide 3% (Fisher Scientific) was used to block endogenous peroxidase activity for 15 min at RT. Sections were preincubated with normal serum for 20 min at RT, then incubated in primary antibody TGF-β1 (Abcam ab66043, 1:200) at 4 °C overnight. After washing in PBST, sections were incubated in biotinylated secondary antibody (Vector Labs, 1:200) for 30 min at RT followed by ABC reagent (Vector labs) for 30 min. Color was developed using ImmPACT™ DAB (Vector Labs). The sections were then counterstained with hematoxylin (Fisher Scientific), dehydrated, and mounted using permanent mounting media (Fisher Scientific). Slides with no primary antibody applied were used as negative controls. The staining results were validated by a blind review performed by a pathologist with extensive experience examining vascular anomalies and immunohistochemistry. Microscopy was performed and images were taken with a Biotek Cytation5 Imaging Reader (Agilent Technologies, California, USA) and Olympus BX43 light microscope with Infinity 3S camera (Olympus, Pennsylvania, USA), respectively.

Fixed mammary glands were washed in ×1 phosphate buffered saline, dehydrated through a series of alcohols and embedded with paraffin (Leica Biosystems, Richmond, IL) under vacuum. paraffin blocks were cut into five micrometer sections on a rotary microtome (Fisher Scientific) and mounted on positively charged slides (Fisher Scientific) for histological evaluation and immunohistochemistry.
For histological evaluations, slides were deparaffinized with xylene and a series of alcohols, stained with Harris’ hematoxylin and eosin (Fisher Scientific), dehydrated, and mounted with permanent mounting media (Fisher Scientific). Digital images were collected using a Zeiss Axio Observer. Z1 inverted microscope, a ×10 objective, and a high-resolution color camera (Carl Zeiss Microscopy).
For quantification of periductal collagen fibers, sections were stained with Gomori’s Trichrome Stain (Richard-Allen Scientific/Thermo Fisher Scientific, REF 87020). In short, deparaffinized samples were fixed with Bouin’s fluid (REF# 88038) and stained with Weigert’s Iron Hematoxylin (REF#s 88028 and 88029) and Gomori’s Trichrome Stain (REF# 88030). Individual dyes in the kit stain the collagen fibers blue, cell nuclei are stained black, while cytoplasm and muscle fibers appear red.

Cochlear slides were washed in dH₂O for 1 min, and then stained for 5 min with the BBC Histo·Perfect™ H&E Stain kit (BBC Biochemical, Mt. Vernon, WA). Slides were rinsed with dH₂O for 1 min, differentiated by BBC Acid wash for 1 min, and rinsed with dH₂O for 1 min; then placed into BBC Blueing solution for 30 sec, and rinsed by dH₂O for 1 min. Next, the slides went into 70% alcohol for 30 sec, stained with BBC Special Eosin II for 1 min, dehydrated with BBC S2*Histo 5 × 20 sec, and cleared in xylene and coverslipped using a permanent mounting media (Fisher Scientific, Pittsburgh, PA). Semi-automated cell counts were made using the MetaMorph imaging system with a Leica DMR microscope, using procedures similar to our previous investigations of the mouse aging auditory system [61 (link), 68 (link), 83 (link)].