Aec substrate kit

Immunohistochemistry (IHC) was performed using the ZytoCHEM-Plus HRP Polymer-Kit (ZYTOMED Systems, Berlin, Germany). Initially, sections were fixed with formalin (Roti®-Histofix 4%, Carl Roth, Germany), followed by blocking of endogenous peroxidase activity by incubation with 3% hydrogen peroxide (Carl Roth GmbH + Co. KG, Karlsruhe, Germany). Tissue sections were then incubated overnight with mouse monoclonal or rabbit polyclonal antibodies against the myofibroblast marker alpha-smooth muscle actin (α-SMA), (Abcam; Cambridge, United Kingdom; ab7817; 1:100); the macrophage marker CD68 (DakoCytomation, Glostrup, Denmark; M0718; 1:50); collagen type 1 (Abcam; ab6308; 1:100); and collagen type 3 (Abcam; ab7778; 1:500). The AEC Substrate Kit (ZYTOMED Systems, Berlin, Germany) was then used for visualization of HRP activity (bound antibody complex) in brown-red color. After immunostaining, sections were counterstained with hematoxylin (Haematoxylin QS, VECTOR, Germany) and mounted in Faramount Aqueous Mounting Medium (DakoCytomation, Glostrup, Denmark).

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.

For immunocytochemical analyses, cells were grown on Superfrost slides (Menzel), dried, fixed with methanol for 5 min, rehydrated in PBS/0.02% Tween 20 for 15 min, treated with 0.9% H₂O₂ for 10 min, rinsed with water, washed with PBS/0.02% Tween 20 for 5 min and incubated with an endogenous avidin/biotin blocking agent (Abcam, ab6421) by following the instructions of the manufacturer. After dilution of the primary antibody in Dako antibody diluent (anti-Sox2: 1:500, anti-PARP-1; cleaved 25 kDa: 1:5000), the slides were incubated with the antibody solution at 4 °C for overnight. Controls were run in Dako antibody diluent in the absence of the primary antibody. Detection of the primary antibody was performed by the ZytoChemPlus (horse radish peroxidase) Broad Spektrum Kit and AEC substrate Kit by following the instructions of the manufacturer (Zytomed, Berlin, Germany). For mounting and visualizing DNA, Immunoselect Antifading Mounting Medium DAPI (4′,6-diamidino-2-phenylindole) (Dianova, Hamburg, Germany) was used. For imaging, AxioCAM MRc5 camera and the AxioVision R 4.5 imaging software (version, Zeiss, Jena, Germany) was used.