Iview dab staining kit

Paraffin-embedded tissue was sectioned in 3 μm slices. After removal of the paraffin and antigen retrieval in citrate buffer (pH 6.0) at 100°C for 5 min, slices were incubated overnight with anti-CD44 antibody (1:100, Abcam, Shanghai, China) at 4°C. Detection was performed in an automated slide staining instrument (Ventana Medical Systems, Tucson, AZ, USA) by using the iView DAB staining kit (Ventana Medical Systems, Tucson, AZ, USA) and the slices were counterstained by hematoxylin.

Paraffin-embedded tissue was sectioned in 3 μm slices. After removal of the paraffin and antigen retrieval in citrate buffer (pH 6) at 125°C for 30 sec, slices were incubated overnight with anti-TMEM26 antibody (Sigma-Aldrich, 1:100) at 4°C. Detection was performed in an automated slide staining instrument (Ventana) by using the iView DAB staining kit (Ventana). The slices were counterstained by hematoxylin and embedded in mounting medium. Staining was scored for area of positive carcinoma cells (area score: 0 = 0%, 1 = 1-9%, 2 = 10-50%, 3 = 51-80%, 4 = 81-100%) and staining intensity (0 = no, 1 = low, 2 = intermediate, 3 = strong staining). These two scores were combined in an IHC score by multiplication and a score of 8 or higher was considered as “high TMEM26” IHC score.
For immunocytochemical staining of TMEM26, cells were grown on Superfrost slides (Menzel) and fixed by using formaldehyde. Anti-TMEM26 reactivity was visualized by using a biotinylated secondary antibody/streptavidin horse peroxidase conjugate-based assay (Zytomed, HRP060) and an AEC substrate kit (Zytomed) by following the manufacturer's instructions.