Anti mouse igg alexa flour 488

Collagenase type II, Elastase, and Exisulind were obtained from Sigma-Aldrich co. (St Louis, MO, USA). PDGF-BB was purchased from R&D systems (Minneapolis, MN, USA). Goat anti-PKG I, rabbit anti-VE-cadherin, mouse anti-osteoponin, rabbit anti-CD31, and goat anti-actin antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit anti-VASP (Ser239), rabbit anti-t-Akt, and mouse anti-p-Akt (Ser473) were purchased from Cell Signaling (Berkely, MA, USA). Mouse anti-calponin antibody was obtained from Sigma-Aldrich co. (St Louis, MO, USA). Mouse anti-α-SMA antibody was purchased from Abcam (Cambridge, MA, USA). The secondary antibodies to each primary antibody were as follows. Anti-mouse IgG HRP conjugated and anti-rabbit IgG HRP conjugated antibodies were obtained from Promega (Madison, WI, USA). Anti-goat IgG HRP conjugated antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-mouse IgG Alexa flour 488 and anti-rabbit IgG Alexa flour 594 secondary antibodies were purchased from Invitrogen (Carlsbad, CA, USA). Alzet osmotic pump was purchased from Durect corporation (Cupertino, CA, USA).

Tissues were fixed overnight in Bouin’s solution (Sigma-Aldrich, HT10132), paraffin-embedded and sectioned at 3 μm. Sections were subjected to hematoxylin-eosin stain and periodic acid–Schiff stain (Merck Millipore, Billerica, MA, USA) following manufacturer’s instructions. For immunofluorescent labeling, sections were first antigen retrieved in boiling 10 mM citrate buffer (pH = 6.0) for 12 minutes. The sections and the spermatozoal spreads were blocked with 10% goat serum (Gibco, Grand Island, NY, USA) and 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA). The following primary antibodies were used for detecting full length dystrophin Dp427 (MANDYS1 3B7; 1:500; Wolfson CIND, OSWESTRY, UK), Dp71 (MANDRA1 7A10; 1:100; Wolfson CIND, OSWESTRY, UK), utrophin (MANCHO3 8A4; 1:100; Wolfson CIND, OSWESTRY, UK), α-tubulin (1:1000; Sigma-Aldrich, T9026), ki67 (1:500; GTX16667; GeneTex, Irvine, CA, USA) and MARK2 (1:100; GTX111783; GeneTex, Irvine, CA, USA). The spermatozoal spreads were also immunofluorescently labeled with MitoTracker® Green (200 nM; Invitrogen, Carlsbad, CA, USA). The following second antibodies were used to provide fluorescent signals: anti-mouse IgG-Alexa Flour 488 (1:1,000; Invitrogen, Carlsbad, CA, USA), anti-rabbit Igs-Alexa Flour 568 (1:1,000; Invitrogen, Carlsbad, CA, USA).