Mach 3 rabbit probe

Antigen-retrieval was performed on a tissue microarray of paraffin-embedded breast tumor cores (US Biomax BR20829) at 95° C for 20 minutes. The microarray slide was rinsed in DI water then incubated with blocking buffer (5% BSA + 5% goat serum in TBS) for 1 hour at room temperature. The slide was then incubated with rabbit anti-periostin antibody (Abcam ab215199, 1:1000) overnight at 4° C. The next day, the slide was rinsed 3 times with TBS and incubated for 30 minutes at room temperature with Biocare’s MACH 3 Rabbit Probe (M3R533 G). Following another three TBS washes, the slide was incubated for 30 minutes at room temperature with Biocare’s MACH 3 Rabbit AP-Polymer (M3R533 G). The slide was then rinsed in TBS and developed using Biocare’s Warp Red Chromogen Kit (WR 806 H) for 8 minutes at room temperature. The slide was washed in TBS, counterstained for 3 seconds with hematoxylin QS, then rinsed in water and TBS. Finally, the microarray slide was dipped in 100% ethanol then xylene and mounted using Biocare’s Ecomount (EM897L). Fluorescent AP signal was imaged using a Nikon Eclipse Ti-E inverted microscope and NIS-Elements software and percent periostin positive area (by pixel) was quantified by thresholding in FIJI.