Armc5 antibody

IHC was performed on serial sections from paraffin-embedded adrenal tissue from selected patients. Slides were deparaffinized in Histoclear (HS-202, National diagnostics, USA) and rehydrated through ethanol gradient. Epitope retrieval was done in Vector Antigen Retrieval Solution (H3300, Vector Labs, USA) at 95°C. ARMC5 antibody (NBP1–94024, Novus, USA) was diluted 1:50 and sections were incubated overnight at 4°C. Detection of the primary antibody was performed using an anti-rabbit antibody coupled to biotin (111–065-144, Jackson Immuno Research Laboratory, USA) followed by streptavidin-HRP amplification (016–030-084, Jackson Immuno Research Laboratory). HRP activity was detected with 3,3’-diaminobenzidinetetrahydrochloride (DAB) (SK-4105, Vector Labs). Negative controls were not used since the tissue itself showed positive and negative ARMC5 staining. Representative pictures of the staining were taken in an inverted light microscope at 5x and 10x objective lenses. Quantification of DAB positive staining was performed using ImageJ software and optical density (OD) was calculated as follows: OD = log(maximum intensity/mean intensity).

Paraffin-embedded normal human brain slides were provided by Alpha diagnostic international (HTS-10301). The slides were deparaffinized in Histoclear (HS-202, National diagnostics) and rehydrated through ethanol gradient. After the epitope retrieval in Vector Antigen Retrieval Solution (H3300, Vector Labs) at 95°C, ARMC5 antibody (NBP1-94024, Novus, USA) diluted 1/50 was incubated overnight at 4°C. The primary antibody was detected using an anti-rabbit antibody coupled to biotin (111-065-144, Jackson ImmunoResearch Laboratory) following by a streptavidin-HRP amplification (016-030-084, Jackson ImmunoResearch Laboratory). The HRP activity was detected with 3,3'-diaminobenzidine tetrahydrochloride (DAB) (SK-4105, Vector Labs). The slides were counterstained with hematoxylin (K8008, Dako).