Dako real envision detection system k5007

IHC was performed according to standardized protocols, of which a detailed description is described in available publications [27 (link),28 (link),29 (link)].
Prepared FFPE microscopic slides were deparaffinized, cleared in xylene and rehydrated in a series of decreasing alcohol solutions ending in TBS. Heat-induced epitope retrieval was performed using a vegetable steamer. The slides were blocked with 5% goat serum after which they were incubated overnight at 4 °C with primary antibody diluted in 1% BSA/TBS/0.1% Tween-20 (antibody details in Supplementary S1). Slides were then rinsed with TBS, incubated with 3% H₂O₂ to block endogenous peroxidase activity and rinsed again with TBS. The next step involved the application of a secondary antibody (Dako REAL EnVi-sion Detection System, K5007, Agilent Technologies, Santa Clara, CA, USA) and incubation at 37 °C for 1 h, followed by a serial TBS wash three times for 5 min. Subsequently, the samples were incubated for 6 min in DAB (3,3-diaminobenzidine-tetrahydrochloride) (Dako REAL EnVision Detection System, K5007, Agilent Technologies, Santa Clara, CA, USA). Slides were counterstained with hematoxylin, embedded and analyzed using the Olympus BX51 microscope (Olympus, Tokyo, Japan). Appropriate positive and negative controls for each antibody were used.

Here, 4 µm sections from formalin-fixed paraffin-embedded (FFPE) tumour samples were immunohistochemically stained with Dako REAL EnVision Detection System K5007 (Agilent, Germany) according to the manufacturer’s protocol against SLC35F2 (anti-SLC35F2 1:100, HPA048185, Atlas antibodies, Bromma, Sweden). Heat-induced epitope retrieval was done in Tris-EDTA Buffer (10 mM Tris Base, 1 mM EDTA, 0.05% Tween20, pH 9.0) at 98 °C for 20 min. A consecutive section was H & E-stained. Immunohistochemical staining was assessed by intensity (no staining (−), weak (+), moderate (++), strong (+++)) of tumour cells. The results were validated and confirmed by a uropathologist.

Protein expression of tumour samples was analysed by immunohistochemical staining with Dako REAL EnVision Detection System K5007 (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s protocol against BCL9L (anti-BCL9L 1:100, HPA049370, Atlas Antibodies, Bromma, Sweden). Briefly, 4 µm sections from formalin-fixed paraffin-embedded (FFPE) tumour samples underwent deparaffinisation and the heat-induced epitope retrieval was done in Tris-EDTA Buffer (10 mM Tris Base, 1 mM EDTA, 0.05% Tween20, pH 9.0) at 98 °C for 20 min. A consecutive section was Hematoxylin–Eosin stained. Immunohistochemical staining was assessed by intensity: (no staining (−), weak (+), moderate (++) and strong (+++)) of the tumour cells. Moreover, the immunohistochemical staining was assessed by the H-Score. The H-Score is calculated by the formula: 3× percentage of strong staining + 2× percentage of moderate staining + percentage of weak staining [49 (link)]. The results were validated and confirmed by a uropathologist.