We detected newly generated neurons derived from two different (transplant and host) sources of NSCs in the thick tissue at the anastomosis; almost all neurons were distributed within 160?m. analyzed using Image J (1.48v, NIH, Bethesda, MD, USA), and some three-dimensional images were made by IMARIS (Bitplane, South Windsor, CT, USA). Fluorescence imaging by confocal microscopy and immunohistochemistry of sectioned preparations Mice were euthanized by an excessive dose of nembutal after in vivo imaging was finished. Fixed frozen blocks and sections of mouse tissues for Rabbit Polyclonal to TAF1 immunohistochemistry (IHC) and fluorescence imaging by confocal microscopy were obtained from Genostaff Co., Ltd (Tokyo, Japan). The ileum along with an anastomosis was fixed with 4?% paraformaldehyde at 4?C for 16?h and was embedded in Cryo Mount 1 (MUTO Pure Chemicals, Co., Ltd., Tokyo, Japan) according to the proprietary procedures. From each block 6-m sections were consecutively cut. Each section was examined with a confocal microscope (Olympus FV1000, Tokyo, Japan). For IHC, each section was washed with PBS to remove the excess compound. Antigen was retrieved by heat treatment at 80?C, 40?min, with sodium citrate buffer at pH 6.0. Endogenous peroxidase blockade with 0.3?% H2O2-methanol, 30?min, was performed, followed by incubation with Protein Block (DAKO Corp., Carpinteria, CA, USA) and avidin/biotin blocking kit (Vector Laboratories, Inc., CA, USA). The sections incubated with mouse monoclonal antibody for PGP9.5 (catalog no. ab8189, 0.4?g?ml?1, Abcam PLC., Cambridge, UK) at 4?C overnight were then incubated with biotin-conjugated goat anti-rabbit Ig (DAKO) diluted 1:600, 30?min, at room temperature and followed STAT3-IN-1 by the addition of peroxidase conjugated streptavidin (Nichirei Biosciences Inc., Tokyo, Japan), 5?min. Diaminobenzidine solution (DAKO) visualized peroxidase activity. The sections counterstained with Mayers hematoxylin (MUTO) were dehydrated and then mounted with Malinol (MUTO). Statistics STAT3-IN-1 Multiple comparisons by one-way analysis of variance (ANOVA) with post hoc Bonferronis test were performed. A value of indicate projections. 100?m In vivo images of STAT3-IN-1 the anastomotic region in MOS-treated YFP mice 2?weeks after NSC transplant Initially we confirmed that enteric neurons were visible in vivo in the terminal ileum, which had a maximal thickness of 50?m at the myenteric ganglion level in an intact Thy1-YFP mouse. This has been previously reported for Thy1-GFP mice [7]. In Thy1-YFP mice treated with MOS (100?M), 2?weeks after gut surgery and NSC transplantation, STAT3-IN-1 yellowish-green fluorescence-positive (YFP [+]) neurons that possibly differentiated from the mobilized (host) NSCs were observed around the knot at the anastomotic site in the thick granulation tissue at a depth of 1C201?m from the serosal surface. Surprisingly yellow-orange PKH26 fluorescence-positive (PKH [+]) neurons that possibly differentiated from the transplanted NSCs were also observed in the same site (Fig.?2a, a-1) in three out of four mice. In one mouse, we could not observe the anastomotic site because of its severe adhesion. As shown in Fig.?2a-1, it seems likely that newly differentiated neurons project their axons to other neurons. Open in a separate STAT3-IN-1 window Fig.?2 Two photon-excited fluorescence microscopy (Two-PM) in vivo images of the anastomotic region in an NSC-transplanted and MOS-treated YFP mouse for 2?weeks. Around the knot at the anastomotic site we obtained an image including newborn neurons differentiated from mobilized (host) NSCs (yellowish axis to a total depth of 1C201?m. indicate nerve cells differentiated from transplanted NSCs. indicate newborn neurons from mobilized NSCs. The area framed by a dotted square in (a) was enlarged into (a-1). a-1 indicate neurons from transplanted NSCs. indicate cell processes connecting neurons, and the indicate presumed cell processes. indicate newborn neurons from mobilized NSCs Confocal microscopy images of the longitudinal ileum section including anastomosis After in vivo imaging with 2PM, the longitudinal ileum section including the anastomosis was observed under a confocal microscope in MOS-treated mice (Oral side of the gut. c-1 correspond to a square 1 in (c) in the granulation tissue. DAB products indicate PGP9.5 (+) ganglionic cells. PKH26 (+) cells were included in PGP9.5 (+) ganglia but YFP (+) cells were not included Immunohistochemistry (IHC) of the longitudinal ileum section including the anastomosis with the anti-PGP9.5 antibody To determine whether the PKH26 (+) aggregates were differentiated neurons from the transplanted NSCs, the immunoreactivity to anti-PGP9.5 antibody was examined (axis depth of 140?m). The other three mice treated with MOS also showed similar results. Open in a separate window Fig.?4 Two-PM images of the anastomotic region in an NSC-transplanted and MOS-treated YFP mouse for 2?weeks. PKH26 fluorescence (+)/YFP fluorescence (+) [PKH26 (+)/YFP (+)] neurons distributed in each of the three areas (b-1, -2, -3) of each of nine field (a-1Cc-3; field size: 310?m??310?m) around the knot at the anastomosis were demonstrated. (indicated nucleus. b A mid-right.