Tyramide alexa fluor 488

Frozen sections of 12 μm thickness were prepared as described above. After blocking with PBS containing 2% normal goat serum, the sections were incubated overnight at room temperature with 1:10,000 diluted anti-ChgH, Hm, or L rabbit IgG and 1:250 diluted anti-ZPb, c5, or AX1 mouse IgG in PBS containing 2% normal goat serum (Table S7). For detection of mouse IgG, the sections were incubated with Goat anti-Mouse IgG Biotinylated (Vector Laboratories, Inc) diluted at 1:1000 for 1 h at room temperature, washed with PBS, and incubated with HRP conjugated ABC regent (VECTASTAIN ABC kit, Vector Laboratories, Inc) for another 30 min. Then the washed sections were incubated with Alexa Fluor 488 tyramide (Invitrogen) for 7 min at room temperature. For detection of rabbit IgG, the sections were incubated with Goat anti-Rabbit IgG Alexa Fluor 555 (Invitrogen) diluted 1:1000 for 30 min at room temperature. After washing with PBS, the sections were sealed in CC mount (Diagnostic BioSystems).

In situ hybridization was performed using DIG-labeled RNA probes and anti-DIG::AP antibody (Roche). Signal was developed using NBT/BCIP (BM Purple, Roche), or Fast Red/HNPP (Roche). Immunocytochemistry was performed using anti-Eve (mouse monoclonal antibody 2B8, hybridoma bank, University of Iowa) as primary, and anti-mouse::POD as secondary antibody (ABC kit, Vector). AlexaFluor 488 tyramide (Invitrogen) was used to give green fluorescent signal. All expression analyses were performed using embryos from uninjected GA-1 strain (WT) or adult GA-1 females injected with double-stranded RNA (ds RNA) of the gene of interest. dsRNA was synthesized using the T7 megascript kit (Ambion) and mixed with injection buffer (5 mM KCl, 0.1 mM KPO₄, pH 6.8) before injection. Used dsRNA concentrations: 200 ng/µl for severe Tc-cad, 7.5 ng/µl for mild Tc-cad, 200 ng/µl for Tc-lgs, 200 ng/µl for Tc-pan, 1 µg/µl for Tc-apc1, 1 µg/µl for Tc-zen1, and 200 ng/µl; 1 µg/µl for Tc-lgs;Tc-zen double RNAi.

Embryos were fixed with 4% paraformaldehyde in PBS overnight at room temperature. The primary antibody (rat anti-GFP IgG, Nacalai tesque or mouse anti-HuC IgG, Santa Cruz Biotechnology) and the secondary antibody (biotin-conjugated anti-rat IgG or biotin-conjugated anti-mouse IgG, VECTOR) were used at a 1∶1000 dilution. Staining was done by using VECTASTAIN ABC kit (VECTOR) with DAB (MUTO PURE CHEMICALS) or Alexa Fluor 488 tyramide (Invitrogen). In the experiment that detects HuC-positive enteric neurons, immnostaining was performed on cryostat sections made from frozen samples mounted in O.C.T. (Sakura Finetek Japan).

Frozen brain tissue from AD subjects was obtained from the Massachusetts Alzheimer’s Disease Research Center (http://madrc.mgh.harvard.edu/) tissue bank. All the study subjects or their next-of-kin gave written informed consent for the brain donation at their respective institutions. Tissue was sectioned on a cryostat at a thickness of 10 µm and fixed for 10 minutes in 4% paraformaldehyde followed by overnight incubation at 4°C with either biotinylated β55 aptamer or its reverse complement, β55rc, used as a control probe. Probes were visualized by reacting with avidin binding complex followed by tyramide signal amplification using AlexaFluor 488 tyramide (Invitrogen, Carlsbad, CA). For slides co-stained with Thioflavin-S, AlexaFluor 594 tyramide (Invitrogen, Carlsbad, CA) was used for visualizing β55 positive plaques in the red channel. Slides were coverslipped and imaged on either an Olympus BX51 fluorescence microscope (Olympus, Tokyo, Japan) or a Zeiss LSM 510 confocal microscope (Carl Zeiss MicroImaging, Jena, Germany).

For histological assessment of human-derived EPCs incorporation in endothelial cells of rat ischemic limbs with the intramuscular injection of the EPCs, we stained 5-μm frozen sections of the rat ischemic adductor muscles with CD31 monoclonal antibody (BD Pharmingen; labeled with Dylight549 IgG, Jackson Immunoresearch Laboratories) and proliferative cell nuclear antigen (PCNA) monoclonal antibody (abcam; labeled with HRP IgG, Molecular Probes and Alexa Fluor 488 Tyramide, Invitrogen). The CD31 antibody and PCNA antibody specifically react with human cells and rat cells, respectively. The numbers of PCNA-positive cells incorporated in CD31-positive endothelial cells (>10 μm) in 10 randomly selected high-power fields (×400) of the same traverse sections were counted with a fluorescence microscope (BZ-9000, Keyence).