Donkey anti mouse igg conjugated to cy3

Samples were fixed in 4% paraformaldehyde and embedded in paraffin before being cut and mounted on polysine glass slides. Those sections were deparaffinized and rehydrated using standard laboratory procedures, then post fixed in 4% paraformaldehyde in phosphate buffered saline for 10 minutes. Heat mediated antigen retrieval was carried out for 20 min in 0.05% citraconic anhydride buffer pH 7.4 for all stainings except for BMP-1 where 10 mM citrate buffer pH 6.0 was used. Then, primary antibodies in 1.25% bovine serum albumin (BSA) were applied (according to Table 2) and incubated overnight at 4°C. Negative controls were made omitting primary antibody, while for ADAMTS-2, ADAMTS-3 and BMP-1, isotype controls were used. After rinsing, secondary antibodies in 12.5% BSA were applied at room temperature for 1.5 hours. The secondary antibodies used were goat anti-rabbit IgG conjugated to Alexa 488 (Invitrogen, Carsbad, CA) and donkey anti-mouse IgG conjugated to Cy3 (Jackson Immuno Research Europe, Newmarket, UK), used at 5.0 and 1.4 µg/ml, respectively. The stained sections were mounted with prolong gold antifade (Invitrogen), containing DAPI for nuclear stain. Imaging was done using an upright Nikon Eclipse E600 microscope equipped with an Olympus ColorView III camera.