Probeon plus

Tissue for RNA in situs were prepared and sectioned according to the LCM protocol. Sections were floated on methanol and mounted on microscope slides (Fisherbrand Probe On Plus). Slides were heated on a slide warmer at 42 °C for overnight. Slides were de-paraffinized in xylene for 10 min twice, followed by a hydration series: 2 times of 100% ethanol, 95% ethanol, 70% ethanol, 50% ethanol, 30% ethanol, and 2 times of DEPC-treated water (2 min for each). Rehydrated slides were incubated in 0.2 M HCl for 10 min, and then washed in DEPC-treated water, 2 times of 2 × SSC (saline sodium citrate), then DEPC-treated water, 5 min each. Slides were then treated with a mixture of 100 mM Tris pH 8.0, 50 mM EDTA pH 8.0 and 1 μg/mL proteinase K at 37 °C for 20 min, followed by a wash in PBS (phosphate buffered saline) for 2 min. Slides were incubated in 2 mg/mL glycine in PBS for 2 min to block proteinase K, and washed in PBS for 30 s twice. Slides were fixed in 4% formaldehyde in PBS for 10 min and washed in PBS for 5 min twice, then dehydrated as follows: 2 times of DEPC-treated water, 30% ethanol, 50% ethanol, 70% ethanol, 95% ethanol and 2 times of 100% ethanol (2 min each). Slides were dried for 15 min in the fume hood.

Livers from aged rats after multiple injections of hPD-MSC therapy were fixed in 10% neutral buffered formalin. After dehydration, fixed tissue was embedded in paraffin, sectioned into 8-μm-thick sections, and secured on slides (Pro-beOn Plus, Fisher Scientific, Pittsburgh, PA, USA). Tissue sections were deparaffinized, incubated with 3% H₂O₂ in methanol for 20 min, and treated with primary antibodies specific for PCNA (ab92552, Abcam, Cambridge, MA, USA) and Ki-67 (ab16667, Abcam, Cambridge, MA, USA) at a 1:100 dilution overnight at 4 °C. Next, sections were rinsed in PBS and treated with biotinylated secondary antibody (DAKO, Carpenteria, CA, USA) for 20 min at room temperature. Finally, the streptavidin-biotin-peroxidase complex was treated for 25 min at room temperature, and peroxidase activity was visualized with AEC+ staining (DAKO, Carpenteria, CA, USA). Negative control sections were incubated with dilution buffer alone. Hematoxylin was used for counterstaining.