Antibody diluent s2022

Formalin-fixed 4 μm sections were deparaffinised with xylene and rehydrated through graded ethanol and demineralised water. Antigens were retrieved by boiling in 10 mM citrate buffer, pH 6.0 in a pressure cooker. Non-specific binding was blocked using Antibody Diluent S2022 (Dako). Consecutive tissue sections were incubated with the following primary monoclonal mouse anti-human antibodies diluted in Antibody Diluent S2022 (Dako): anti-TF (No. 4509, American Diagnostica), anti-Epithelial Membrane Antigen (MUC1) (clone E29), anti-CD68 (clone KP1) and anti-CD31 (Clone JC70A) from Dako. After blocking endogenous peroxidases, staining was visualised using a peroxidase-labelled polymer conjugated to goat anti-mouse immunoglobulins and 3,3′-diaminobenzidine (K4007, Mouse EnVision⁺ kit, Dako). Sections were decolourised, counterstained with haematoxylin (1 : 1 dilution with demineralised water; Merck, Darmstadt, Germany), decolourised again, dehydrated and finally permanently mounted.
Mouse IgG1 (BD Biosciences) and omission of the primary antibody served as specificity controls. Normal pancreatic islet cells, tonsils and ductal breast cancer cells served as positive control for TF, CD68 and MUC1 staining, respectively. No separate control was prepared for CD31 staining.

CDX2 was detected in paraffin sections of LCNEC tissue. Four-micrometer sections were prepared for tissue slides. Antigen retrieval was performed at 95°C for 10 minutes in a microwave with Target Retrieval Solution (pH 9.0) S2367 (DAKO, Glostrup, Denmark) after deparaffinization. Followed by a cooling down period at room temperature for 20 minutes, then treatment with 3% hydrogen peroxide for 15 minutes at room temperature. After the treatment with TBST buffer S3006 (DAKO), Protein Block, Serum Free Solution X0909 (DAKO) was used to block non-specific binding incubate for 10 minutes at room temperature. Staining with anti-human CDX2 clone DAK-CDX2 M3636 (DAKO) with diluents using Antibody Diluent S2022 (DAKO), 1:100, was performed overnight at 4°C. After the treatment with TBST buffer S3006 (DAKO), Histofine Simple Stain MAX-PO (Nichirei, Tokyo, Japan) for CDX2 was applied and incubated for 30 minutes at room temperature. The substrate used DAB, and nuclear staining used hematoxylin. Negative controls used Negative Control Mouse IgG1 X0931 (DAKO) instead of the primary antibody. Immunohistochemical (IHC) staining was evaluated as previously described.^{26 (link)}All slides were interpreted by two independent observers in a blinded fashion. For evaluation reliability, two independent assessors estimated the staining positivity of two serial sections.