Anti mouse 546 a11030

Adult samples were fixed in 4% paraformaldehyde in 0.1 mol/L phosphate buffer overnight and decalcified with 0.5 mol/L ethylenediaminetetraacetic acid for 3 days to 1 week at 4°C. Seven micron paraffin sections were prepared. Sections were treated for antigen retrieval with 0.01 mol/L citrate buffer (pH 6.0) by microwaving for 6 min. Immunohistochemistry was performed according to Jiang et al. (1998) using the following antibodies (1:200 dilution): β‐catenin (Sigma; C2206, St Louis, MO); PCNA (Chemicon; CBL407, Billerica, MA); NCAM and tenascin‐C (Chuong and Chen 1991). Secondary antibodies were either biotinylated anti‐mouse IgG or anti‐rabbit IgG (Vector Laboratories, Burlingame, CA, USA; 1:200 dilution). The tertiary antibody was streptavidin (Vector Laboratories, 1:200 dilution). An AEC substrate kit (Vector Laboratories) was used to develop the staining. Hematoxylin was used to perform faint counterstaining. BrdU staining was performed according to Wu et al. (2004). For BrdU (BD; 347580; 1:200 dilution)/β‐catenin or PCNA/β‐catenin double staining, secondary antibodies Alexa Fluor anti‐rabbit‐488 (A11008) and anti‐mouse‐546 (A11030) from Invitrogen (Grand Island, NY) were used at a 1:200 dilution. 4’,6‐Diamidino‐2‐phenylindole (DAPI) was used to visualize the nuclei. Stained sections were imaged with a Zeiss 510 confocal microscope.