Formalin phosphate

Explanted bladder specimens encompassing the entire thickness of the bladder were isolated immediately following euthanasia and processed as previously described [15 (link)]. Briefly, specimens were fixed in a 10% buffered formalin phosphate (Fisher Scientific, Inc.) solution followed by a series of graded ethanol exchanges then embedded in paraffin (Fisher Scientific). Embedded tissues were sectioned onto glass slides at a thickness of 10 μm using a RM2125 RT Microtome (Leica) onto glass slides and subjected to staining with Masson’s Trichrome (Sigma–Aldrich Corp.) reagent. The paraffin was removed from tissue containing slides using a hot plate at 62 °C for 6 min and was followed by treatment with xylenes, graded ethanol washes and DI water. Slides were placed in Bouin’s solution (Sigma–Aldrich Corp.) for approximately 15 min then rinsed under running tap water. The samples were then stained 5 min with Hematoxylin and rinsed with running water and subsequently stained with Scarlet-Acid Fuchsin (Sigma–Aldrich Corp.). Slides were rinsed again with DI water and placed into a mixture of PTA/PMA, followed by Aniline Blue solution and a 1% acetic acid wash. Finally, slides were placed in 95–100% ethanol and rinsed in xylene. Following air drying, a coverslip was placed over the specimen sample and secured with Permaslip (Alban Scientific Inc).

For LacZ staining, inguinal mammary glands and mammary tumors were fixed at room temperature for 30–45 minutes in 4% paraformaldehyde (Electron Microscopy Sciences). Tissues were either transferred to 30% sucrose/PBS (w/v) for frozen sections, or incubated for 18–24 hours in PBS containing 4 mM potassium hexacyanoferrate (III) (Sigma-Aldrich), 4 mM potassium hexacyanoferrate (II) trihydrate (Sigma-Aldrich), 2 mM MgCl₂ (Fisher Scientific), 0.2% NP-40 (Fisher Scientific), 0.1% sodium deoxycholic acid (Fisher Scientific) and 1 mg/ml Xgal (Invitrogen). Stained tissues and whole mounts were post-fixed with 10% formalin-phosphate (Fisher Scientific). Senescence-associated β-gal staining was carried out on mammary gland cryo-sections as described for the Senescence β-galactosidase Staining Kit (Cell Signaling). Sections were stained overnight (12–15 hours) in a non-humidified 37°C incubator, counter-stained with Eosin, and dehydrated with a graded ethanol/xylene series before mounting with Permount Toluene Solution (Fisher Scientific).