Biotin conjugated anti rabbit goat antibody

Seeds kept at −80°C were hand dissected whilst frozen and immediately fixed in 0.1 M phosphate buffer pH 7.2 plus 4% paraformaldehyde, 0.1% glutaraldehyde. Samples were then dehydrated in an ethanol series and embedded in Paraplast (Sigma). Sections 8 μm thick were attached to sylanized glass slides. Slides were deparaffinised with xylene (Dimethyl-benzene) and then rehydrated in an ethanol series. Endogenous peroxidase activity was deactivated by incubation in 0.3% hydrogen peroxide for 20 min. Sections were then blocked with 2% normal donkey serum for 2 h at room temperature (RT) and reacted with antiBETL1 or antiBETL2 antisera, or the corresponding pre-sera at 1:500 dilution. Reacted primary antibodies were detected with biotin-conjugated anti-rabbit goat antibody diluted 1:750 (Sigma) and then with Extravidin-peroxidase (Sigma) solution at 1:800 dilution. Finally, positive reaction was developed using SIGMAFAST™ DAB with Metal Enhancer (Sigma) until a gray-black precipitate was clearly visible on the sera-reacted slides. The sections were stained post-detection with 0.025 % Azure B in phosphate buffer pH4 for 3 min, washed in abundant distilled water, mounted in DEPEX and photographed as described in Muñiz et al. (2006 (link)).

Seeds kept at −80°C were hand dissected whilst frozen and immediately fixed in 0.1 M phosphate buffer pH 7.2 plus 4% paraformaldehyde, 0.1% glutaraldehyde. Samples were then dehydrated in an ethanol series and embedded in Paraplast (Sigma). Sections 8 μm thick were attached to sylanized glass slides. Slides were deparaffinised with xylene (Dimethyl-benzene) and then rehydrated in an ethanol series. Endogenous peroxidase activity was deactivated by incubation in 0.3% hydrogen peroxide for 20 min. Sections were then blocked with 2% normal donkey serum for 2 h at room temperature (RT) and reacted with antiBETL1 or antiBETL2 antisera, or the corresponding pre-sera at 1:500 dilution. Reacted primary antibodies were detected with biotin-conjugated anti-rabbit goat antibody diluted 1:750 (Sigma) and then with Extravidin-peroxidase (Sigma) solution at 1:800 dilution. Finally, positive reaction was developed using SIGMAFAST™ DAB with Metal Enhancer (Sigma) until a gray-black precipitate was clearly visible on the sera-reacted slides. The sections were stained post-detection with 0.025 % Azure B in phosphate buffer pH4 for 3 min, washed in abundant distilled water, mounted in DEPEX and photographed as described in Muñiz et al. (2006 (link)).