Fig. 2

The ADP2 IHC assay detects a range of ADAM8 levels with high specificity and reproducibility. A ADP2 staining is specific for ADAM8 in competition analyses. ADP2 was pre-incubated overnight at 4 °C at 1:1000 dilution in the absence or presence of 1×, 10× or 100× molar equivalents of purified recombinant human ADAM8 protein (rHuA8), and used in IHC of HEK-A8-2D and MB-231-3D cells. IgG2b was employed as isotype control. ADP2 staining was reduced in a dose-dependent manner in the presence of rHuA8, demonstrating the assay’s specificity for ADAM8. B ADP2 has excellent range and linearity of ADAM8 detection. IHC was performed using slides of the breast CCM and a range of ADP2 dilutions from 1:50 to 1:120,000 under the optimized ADP staining conditions vs isotype control IgG2b at 1:50. ADP2 detected both very low and very high levels of ADAM8 in a dose-dependent manner. C ADP2 detects ADAM8 in PDX tumor tissue samples at levels that are within the range of the CCM. IHC was performed using ADP2 at dilutions of 1:50, 1:100 and 1:500 vs isotype control IgG2b at 1:50 and ADAM8-expressing TNBC PDX samples 5998, 3561, and 4849 vs the breast CCM. All 3 PDX samples displayed strong ADAM8 staining that was in the range of that seen in the breast lines of the CCM, indicating the latter is suitable for scoring of more complex tissue samples. Representative images of staining with ADP2 1:50 dilution, which was identified as optimal for tissue sample analysis, are shown. D ADP2 reproducibly detects ADAM8 in PDX tumor tissue samples. Consecutively cut slide sets of each PDX were processed on different days (3–4 days apart), and demonstrated equal staining, indicating the reproducibility of the ADP2 IHC assay. Representative images of PDX3561 are shown. Images are at 40× magnification. MB-231, MDA-MB-231