Cathepsin B Immunofluorescence in db/db Mice

The paraffin-embedded tissue sections from vehicle or metformin treated db/db mice were used for staining. After deparaffinization (Xylene (twice, 5 min), 100% ethanol (twice, 5 min), 95% ethanol (twice, 5 min), 70% ethanol (once, 5 min), 50% ethanol (once, 5 min), and type one water, the tissue was boiled in 10 mM sodium citrate buffer (Vector labs) for 20 min. After a PBS wash, blocking was performed with 2.5% normal horse serum (Vector labs) at room temperature. Next, the tissue sections were incubated with cathepsin B antibody ((1:500) Cell Signaling Tech, 3383) for 2 h and incubated with a fluoresceine-labeled horse anti-rabbit secondary for 1 hr. After washing with PBS, slides were mounted using Vectashield anti-fade mounting media. Fluorescence images were captured using an Eclipse Ti2 by Nikon Instruments Inc., Melville, NY, USA.