Dako envision system hrp

Five μm formalin-fixed paraffin-embedded (FFPE) liver sections were deparaffinized and rehydrated in xylene and graded alcohol series. Antigen retrieval was performed with Tris-EDTA buffer (Merck) or with citrate buffer (Dako Target Retrieval Solution, Agilent, Santa Clara, CA, USA) (Supplementary Table 2). Non-specific immuno-staining was prevented by 1 hour incubation in PBS containing 5% Bovine Serum Albumin (Merck) ± 5% Normal Goat Serum (Thermo Fisher Scientific, Waltham, MA, USA). Thereafter, sections were incubated with primary antibody for 1 hour at 37C (Supplementary Table 2). After washing, sections were incubated with species-specific secondary antibodies (Dako EnVision+ System HRP, Agilent). Immunohistochemical detection was performed with liquid 3,3′-diaminobenzidine (DAB) chromogen and substrate buffer (Dako, Agilent), and was followed by Mayer’s hematoxylin counterstaining. For immunofluorescence staining, tyramide-fluorophore amplification was performed sequentially with different fluorochromes (Alexa-488, 555, 594 or 647, Atto-425, Thermo Fisher Scientific or Invitrogen, Waltham, MA, USA) for each antibody as previously described [52 (link)]. Sections were mounted with DAPI containing media or counterstained with Hoechst (Merck) and mounted with Dako fluorescence mounting medium (Dako).

GLI1, p-AKT, cyclin D3, CDK4, and Ki67 staining were performed. Briefly, resected tumors from xenograft mice were fixed in 10% buffered formalin, embedded in paraffin, and mounted on slides. After deparaffinization and rehydration, mouse tumor sections were incubated in 3% hydrogen peroxide to suppress endogenous peroxidase activity. Antigen retrieval was achieved by microwaving the sections in 10 mM sodium citrate (pH 6.0). To block the sections, 10% goat serum was then used. Human GLI1, p-AKT, cyclin D3, CDK4, and Ki67 (Abcam) antibodies were incubated with the mouse tumor sections overnight at 4 °C. Detection was performed with the Dako IHC kit (Dako EnVision+ System, HRP, Agilent technologies). Slides were stained with 3,30-diaminobenzidine (Sigma-Aldrich), washed, counterstained with hematoxylin (Sigma-Aldrich), dehydrated, and mounted. Images of each slide were taken using an inverted microscope for data analysis.

Five μm formalin-fixed paraffin-embedded (FFPE) liver sections were deparaffinized and rehydrated in xylene and graded alcohol series. After a 1 hour blocking step in 5% BSA (Merck, Darmstadt, Germany) ± 5% normal goat serum (Thermo Fisher Scientific, Waltham, MA, USA), sections were incubated at 37C with primary antibody for 1 hour (S1 Table in S1 File) followed by species-specific secondary antibodies incubation (Dako EnVision+ System HRP; Agilent, Santa Clara, CA, USA) and 3,3′-diaminobenzidine (DAB) chromogen detection. Digital quantification of staining was assessed on x20 magnification scanned sections by using the image analysis tool Author version 2017.2 (Visiopharm, Hørsholm, Denmark) as previously described [8 (link)].

Brain samples were fixed in 10% neutral buffered formalin. Following fixation, the tissues were dehydrated, cleared, and embedded in paraffin. Sections from the brain paraffin blocks were cut to a thickness of 4 μm. H&E staining was performed using Dako CoverStainer (Agilent, Santa Clara, CA, USA). To confirm the expression of neuroinflammation marker proteins, IHC was performed. The brain sections were stained with antibodies against BDNF, COX2, and GFAP (1:500; Abcam, Cambridge, UK) primary antibodies, and the labeled polymer Dako EnVision + System-HRP (Agilent) was used according with the manufacturer’s instructions. After staining, the brain sections were scanned with a Pannoramic SCAN II scanner (3DHISTECH Kft., Budapest, Hungary). Neuroinflammation markers in the hippocampus were quantified using ImageJ software version 1.53a (NIH, Bethesda, MD, USA).

Tissue samples embedded into paraffin blocks were cut into 4-μm sections. After deparaffinization and rehydration, sections were microwaved in 10 mM sodium citrate buffer for antigen retrieval and stained with anti-BEX4 antibodies (Abcam) at room temperature for 1 h. DAKO EnVision + System, HRP (DAKO) was used for visualization of immunoreaction. Then, sections were stained with Mayer’s hematoxylin, dehydrated and photographed under light microscope. BEX4 protein expression level was graded as low, moderate or high depending on the staining intensity. Association between BEX4 expression and the clinicopathological parameters of OSCC patients was evaluated by Chi-square test or Fisher’s exact test.