Alexafluor 555 goat anti rabbit igg secondary antibody

For in situ whole mount IHC visualization of MMP13a protein expression in the caudal fin in response to tamoxifen, an IgG rabbit polyclonal anti-MMP-13a (hinge) antibody for detecting both proenzyme and cleaved active MMP was used (Anaspec, Inc., Fremont, CA, Cat. No. 55114). The manufacturer’s protocol was adapted with minor changes. Briefly, animals were fixed in 4% paraformaldehyde in PBS at 4 °C overnight, then rinsed 3 times in PBSTx for 10 minutes each, then blocked in blocking buffer 1 (PBSTx with 5% BSA, 5% NGS, 0.1% DMSO) for 6 hours at 4 °C. Primary antibody was applied overnight in blocking buffer 2 (PBSTx with 0.5% BSA, 0.1% DMSO) with a 1:500 dilution at 4 °C. Primary was rinsed off with PBSTx 2 × 5 minutes, then 4 × 30 minutes. AlexaFluor 555 goat anti-rabbit IgG secondary antibody (1:5000 dilution, Molecular Probes, Eugene, OR) was applied in blocking buffer 2 at room temperature for 2 hours. Secondary was washed off with PBSTx 2 × 5 minutes, then 4 × 30 minutes. Stained embryos were then visualized on an inverted Zeiss Axiovert 200M epifluorescence microscope using a Zeiss Axiocam HR camera (Carl Zeiss, Oberkochen, Germany).