Cwk f12

Immunohistochemistry was performed with antibodies against BCL-2 (M0887, Dako, Glostrup, Denmark), BCL-XL (SC7195, Santa Cruz Biotechnology, Santa Cruz, CA, USA), BCL-W (4G12E6, Novus biologicals, Bio-Techne, UK), MCL-1 (A3534, Dako) and ERβ (CWK-F12, 1:160, Developmental Studies Hybridoma Bank, Iowa, USA) on paraffin-embedded tissue sections after antigen retrieval (pH 6,pH 9, pH 9, pH 6, and pH 9, respectively). Staining was visualized using HRP-labeled secondary antibodies (Dako) and 3,3′-diaminobenzidine (Sigma Aldrich, St Louis, MO, USA). Appropriate positive and negative controls were performed for each staining. The cases were stained and scored on a tissue micro-array (TMA). The TMA contained a total of 84 HL cases with 67 cases of NS HL, 7 cases of MC, 1 NOS HL, and 9 NLPHL. Patient material was acquired in accordance with international regulations and professional guidelines (the Declaration of Helsinki and the International Conference on Harmonization Guidelines for Good Clinical Practice). This project has been approved by the internal review board of the UMCG under RR#201800551.

IHC was performed on FFPE tissue according to standard protocols, with appropriate positive and negative controls. FFPE tissue of randomly selected DLBCL cases was used. Surgical rest material falling under the “code for proper use” was used and was approved under RR202100080 (Local Testing Committee Pathology). Sections were stained with anti-ERβ (CWK-F12, 1:160, Developmental Studies Hybridoma Bank, Iowa, USA) after antigen retrieval with 10 mM TRIS/1 mM EDTA pH9 for 15 min at 120 °C, for an hour at room temperature.