Impress secondary antibody

Paraffin-embedded slides were heated on 70°C heat blocks, deparaffinized in xylene, and then hydrated in a series of alcohols (100%, 90%, 70%) for a period of 5 minutes each. After boiling slides in 10mM Tris, 1 mM EDTA (pH 9.0) for 20 minutes, slides were blocked with 2.5% horse serum and primary antibodies were added to the slides overnight at 4°C. After washing in 1X PBS, secondary antibody was added (Impress secondary antibody, Vector Labs, Burlingame, CA) for thirty minutes. DAB (Cell Signaling, Danvers, MA) was added for a period of approximately 1 minute, Hematoxylin was used as a counterstain and the slides were dehydrated in alcohols before mounting. Anti-BZLF1 antibody (BZ.1 clone, Santa Cruz SC53904) was used at a 1:200 dilution, K10 antibody (Biolegend catalogue #905404) was used at 1:4000. All dilutions were in 2.5% Normal Horse Serum (Vector Labs, Burlingame, CA). EBER in situ hybridization studies were performed using the PNA ISH Detection Kit (DakoCytomation) according to the manufacturer’s protocol as previously described [58 (link)].

5 μm thick sections were cut with a microtome (Surgipath), mounted, hydrated in xylene before immersion in descending alcohol concentrations and distilled water. Antigen retrieval was performed when necessary (Table S3). Slides were washed in phosphate buffered saline (PBS), immersed in 1% hydrogen peroxide diluted in methanol (1% H₂O₂/MeOH) for 10 min and washed with PBS. Unspecific binding was blocked with either 2.5% of normal horse or goat serum (Vector laboratories) added for 30 min. The primary antibody was diluted in 1% bovine serum albumin (BSA) in PBS and incubated overnight at 4°C in a humid chamber (Table S3) followed by washing in PBS then incubating with Impress secondary antibody (Vector laboratories) for 30 min. Impress anti-Rabbit, anti-Rat or anti-Mouse was used depending on the species of the primary antibody. Vector® chromagen DAB, SG or VIP was used to visualize antibody complexes. Slides were mounted in DPX and left drying overnight at 65°C. Isotype control antibodies were used at the same concentrations and conditions.