Avidin biotin blocking buffer

Tumors or enlarged organs were removed during necropsy, fixed in 10% formalin at room temperature and embedded in paraffin. Sections were stained with hematoxylin–eosin and analyzed by a pathologist. Sections of paraffin-embedded tissues (5 μm) were deparaffinized in xylene (2 × 5 min) and then rehydrated to distilled water using graded alcohols. Antigen was retrieved by steaming the slides for 20 min and then cooling for 20 min in either 1 mM EDTA, 0.05% Tween 20, pH 8 (for AID and BCL6), or 10 mM acid citric, 0.05% Tween 20, pH 6.0, for all other antigens. Blockings were as follows: 0.3% H₂O₂ for 10 min for endogenous peroxidase, avidin/biotin blocking buffer (#SP2001, Vector Laboratories, Burlingame, CA) for 15 min and/or 3% normal goat serum and 1% bovine serum albumin for 60 min at room temperature. Carbo-free blocking buffer (#SP5040, Vector Laboratories) was used prior to PNA staining for 60 min at room temperature. Sections were incubated with PNA (Vector Laboratories) or primary antibodies (Supplementary Table S1) for 60 min at room temperature or overnight at 4°C, followed by biotin-conjugated secondary antibodies and detection with Vectastain ABC Kit (PK-6100, Vector Laboratories). Peroxidase activity was developed using ImmPACT NovaRED HRP substrate (Vector Laboratories).