Alexa 546 conjugated goat anti mouse igg

The CNS was dissected from flies in PBS, and fixed in 4% paraformaldehyde in PBS for 60 min. Immunostaining was carried out as described previously16 (link), using the following antibodies at the indicated dilutions: rabbit anti-GABA diluted at 1:500 (Sigma), rabbit anti-5-HT at 1:500 (Sigma), chicken anti-GFP at 1:1,000 (Abcam), rabbit polyclonal anti-GFP at 1:1,000 (Molecular Probes) and mouse monoclonal nc82 at 1:20 (DSHB, University of Iowa, IA). The secondary antibodies used were as follows: Alexa647-conjugated goat anti-mouse IgG at 1:200, Alexa546-conjugated goat anti-rabbit IgG at 1:200, Alexa488-conjugated goat anti-chicken IgG at 1:200, Alexa488-conjugated goat anti-rabbit IgG at 1:200 and Alexa546-conjugated goat anti-mouse IgG at 1:200 (all from Invitrogen). Stacks of optical sections were obtained with a Zeiss LSM 510 META confocal microscope and were processed with Image J software.

Rat mAb against mouse laminin α1 (5B7-H1), rat mAb against mouse laminin α5 (M5N8-C8), and rabbit polyclonal antibody (pAb) against Velcro (ACID/BASE coiled-coil) peptides were produced in previous studies (Manabe et al, 2008 (link); Sato-Nishiuchi et al, 2012 (link)). The following antibodies and reagents were obtained commercially: rat anti-laminin-γ1 mAb (Millipore); rabbit anti-laminin pAb, BSA, and heparin (Sigma-Aldrich); mouse anti-CDX2 mAb (Biocare); rabbit anti-OCT4 pAb (Santa Cruz Biotechnology); rat anti-integrin αV mAb (RMV-7), hamster anti-integrin β1 mAb (Ha2/5), and hamster anti-integrin β3 mAb (2C9.G2) (BD Biosciences); Alexa 488–conjugated goat antirabbit IgG, Alexa 546–conjugated goat antirat IgG, Alexa 405–conjugated goat antirabbit IgG, Alexa 488–conjugated goat antirat IgG, and Alexa 546–conjugated goat antimouse IgG (Invitrogen); bovine types I and IV collagens (Nippi Inc.); recombinant human FGF4 (Peprotech); and PermaFluor (Thermo Shandon). Mouse EHS laminin was prepared from a mouse EHS tumor as described (Murayama et al, 1996 (link)). Fibronectin was purified from human plasma by gelatin affinity chromatography as described (Sekiguchi & Hakomori, 1983 (link)).

After mouse brains were dissected, they were immediately fixed in a 10% buffered formalin solution. Sections (3 μm) were then deparaffinized, heated in a microwave for 15 min in a 10 mM citrate buffer (pH 6.0), and incubated overnight with the following primary antibodies: anti-GFP (1:200; D5.1, Cell Signaling Technology), anti-NeuN (1:100; MAB377, Millipore), anti-GFAP (1:1,000; ab53554, Abcam), and anti-Olig2 (1:250; MABN50, Millipore). For immunohistochemistry, the samples were incubated with a secondary antibody labeled with a polymer as part of the Envision+ system containing horseradish peroxidase (Dako Cytomation, Glostrup, Denmark). Photographs of immunohistochemically stained sections were captured using an optical microscope (BX51, Olympus). For immunofluorescence, the samples were incubated for 1 h with the following secondary antibodies: Alexa 488-conjugated goat anti-rabbit IgG (1:1,000; Invitrogen), Alexa-546-conjugated goat anti-mouse IgG (1:1,000; Invitrogen), and biotinylated horse anti-goat IgG (1:500; BA-9500, Vector Laboratories). Biotinylated secondary antibodies were visualized using Alexa-594-conjugated streptavidin (1:1,000; Invitrogen). Nuclei were stained with Hoechst 33342 (1:400; Invitrogen). Immunofluorescence-stained sections were mounted with ProLong Gold (Invitrogen) and then imaged with a fluorescent microscope (BZ-X710, Keyence, Osaka, Japan).

We collected femur specimens on day 14 after surgery for histological analysis. To do so, animals were euthanized, femurs removed and separated from soft tissues, and samples were then fixed in 4% PFA in 0.1 M PBS, demineralized with 10% ethylenediaminetetraacetic acid, embedded in paraffin, cut into 5-μm-thick sections, and Hematoxylin and Eosin-stained (HE). Empty lacunae were detected in femur sections following HE staining, and the percentage of empty lacunae was calculated relative to total (empty + undamaged) lacunae.
For immunohistochemistry, sections were subjected to microwave treatment for 10 min in 10 mM citrate buffer solution (pH 6.0) for antigen retrieval, as described^{40 (link)}. After blocking 1 h with 3% BSA in PBS, sections were stained using mouse anti-P. gingivalis mAb (D376-3 1:100 Medical & Biological Laboratories Co.,LTD, Tokyo, Japan), followed by Alexa546-conjugated goat anti-Mouse IgG (#A-11030 1:200; Invitrogen, Carlsbad, CA, USA). Nuclei were visualized by DAPI (#D1306 1:750; Wako Pure Chemicals Industries, Osaka, Japan).