Ki 67 ncl ki67p

After photographing the wound on day 14, skin tissue including the wound area was excised for pathological examination. The specimens were fixed in 10% formalin for 24 h. The sections were processed routinely and stained with hematoxylin and eosin (HE). By microscopic examination using the HE-stained samples, the thickness of the granulation tissue and the regeneration of the epithelium at the skin defect were measured.
As well as HE staining, some of the specimens obtained from animals in two groups (Untreated RU and ASCs spheroids NS for RU groups) were used for immunohisitological examination using CD31 (DIA-300; DIANOVA GimbH, Hamburg, Germany) and Ki-67 (NCL-Ki67p; Leica Biosystems, Newcastle, UK) antibodies. Briefly, for both stains, antigen retrieval was performed in paraffin-embedded sections with 10 mM citrate buffer for 10 min. Thereafter, 3% H₂O₂ was used to inactivate endogenous peroxidase, and Blocking One (Nacalai Tesque, Kyoto, Japan) was used to block nonspecific binding reactions. The sections were then incubated with primary and secondary antibodies. The sections were treated with 3, 3′‐diaminobenzidine‐4HCl (DAB) and Mayer's hematoxylin nuclear counterstain as per the usual protocols. Microscopic images were taken of five random fields from each slide at high power fields (×400). The positive cells were counted and the average value was calculated.

Immunohistochemistry was performed as previously described (Taulli et al., 2009 (link)) with the specified antibodies. Ki67 (#NCL-Ki67p) was from Leica Biosystems (UK); MyoD (#M3512) was from Dako (Denmark); Myogenin (DSHB (Iowa City, IA) Hybridoma Product F5D was deposited by Wright, Woodring E.); Pax7 (DSHB Hybridoma Product PAX7 was deposited by Kawakami, Atsushi); eMHC (DSHB Hybridoma Product F1.652 was deposited by Blau, H.M.); P-Met (#3126) and PDGFRα (#3164) were from Cell Signaling Technology (Danvers, MA); Met (#18–7366) was from Invitrogen. Fiber cross-sectional areas were measured using ImageJ software (rsb.info.nih.gov/ij). Statistical analyses were performed using Microsoft Excel software, with a moving average (period 4) trendline.

In TRAMP PCa, immunostaining for apoptotic (cl-caspase-3, Cell Signaling Technology) and proliferating (Ki67, NCL-Ki67p, Leica Biosystems, Buffalo Grove, IL) cells was performed using rabbit polyclonal and biotinylated goat anti-rabbit IgG secondary antibodies (Vector Laboratories, Burlingame, CA) as previously described [50 (link)]. Blood vessel density was determined by immunostaining for CD31 using a goat polyclonal antibody (M20; Santa Cruz Biotechnology) and a biotinylated rabbit anti-goat secondary antibody. Immunostaining for Mcl-1 (S-19) was performed using a 1/50 dilution of rabbit polyclonal and biotinylated goat anti-rabbit IgG secondary antibody. Immunostaining for γH2AX (clone JBW301# 05-636; EMD Bioscience) was performed using a 1/200 dilution of mouse monoclonal and the Vector Mouse on Mouse Peroxidase Kit (Vector Laboratories) following the manufacturer's instructions. The number of cleaved caspase-3, Ki67, γH2AX positive cells and CD31 positive vessels were determined for 1198, BA, 1198 + BA, and vehicle controls (n=3-5 each group), as previously described [51 (link)]. For negative controls, we used the same concentration of mouse or rabbit IgG (Santa Cruz Biotechnology) instead of specific primary antibodies, resulting in lack of immunostaining.