Alexa fluor 647 conjugated anti mouse secondary antibody

We performed indirect immunofluorescence analysis after miRNA in situ HCR. After HCR detection, but prior to Hoechst staining, washed sections were incubated in blocking solution (1:400 unlabeled affinity purified Fab fragment donkey anti-mouse IgG (H + L) and 5% donkey serum in TBS: 100 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20). Sections were then incubated with mouse anti-AP2α^{38 (link)} (Developmental Studies Hybridoma Bank) 1:1000 in TBS at room temperature for two hours. The sections were washed in TBS 3 times, then incubated with Alexa Fluor 647-conjugated anti-mouse secondary antibody (1:1000, Jackson Immunoresearch) for one hour at room temperature. Sections were washed in TBS 3 times and mounted in Aqua-Poly/mount.

Proteins (5 μg) were resolved on 12 % polyacrylamide gel under denaturing conditions and transferred onto a polyvinylidene difluoride membrane. Membranes were blocked with phosphate buffered saline (PBS) tween (0.2 % Tween-20; Sigma-Aldrich) and 5 % of milk. Blots were incubated overnight at 4 °C with the rabbit anti-ADAMTS-4 (1/5000; AbCam, Cambridge, UK) or the rabbit anti-ADAMTS-5 (1/1000; AbCam) primary antibodies diluted in PBS-tween containing 5 % of bovine albumin serum (BSA). After a 2-h incubation at room temperature (RT) with the peroxidase-conjugated anti-rabbit secondary antibody (1/2000; GE Healthcare life sciences, Uppsala, Sweden), proteins were revealed with an enhanced chemiluminescence ECL-Plus kit immunoblotting detection system (GE Healthcare life sciences) and visualized using Storm^TM FluorImager system. Mouse anti-β-actin (1/5000; Sigma-Aldrich) was used as a loading control and visualized by Alexa fluor 647-conjugated anti-mouse secondary antibody (1/1000; Jackson ImmunoResearch laboratories Inc., West Grove, PA, USA).