Citrate buffer ph 6

Immunohistochemical (IHC) analysis was performed using FFPE tissues from 265 patients on tissue microarrays (TMA). TMA were constructed using 0.6 mm FFPE tissue cores punched from each donor block. To overcome tumor heterogeneity, three representative cores were selected from each tumor. 5μm thick TMA sections were deparaffinized and pretreated in citrate buffer pH 6.0 (Dakocytomation, Carpenteria, CA) for 20 minutes using an ordinary vegetable steamer. Slides cooled 20 minutes and were incubated 10 minutes with 3%H₂O₂ to quench endogenous peroxidase activity. Blocking was performed using serum-free protein block, Dakocytomation (Carpenteria, CA) for 20 minutes. Anti-CD47 polyclonal (Atlas, Stockholm Sweden) was added at 1:50 dilution to each section and incubated 60 minutes at room temperature. Rabbit IgG (Dakocytomation, Carpenteria, CA) was incubated as our negative isotype matched control. Labeled streptavidin biotin (LSAB2) reagents Dakocytomation (Carpenteria, CA) were used according to the manufacturer’s instructions followed by a 5 minute incubation with 3,3′-diaminobenzidine (DAB)+ Dakocytomation (Carpenteria, CA). Sections were counterstained with hematoxylin and cover slipped.

For visualization of the VP1 major capsid protein of HPyV-6 in formalin-fixed paraffin-embedded tissue samples, slides were deparaffinized and rehydrated. After antigen retrieval with citrate buffer, pH 6.0 (Dako), and a wash with phosphate-buffered saline (PBS), peroxidase-blocking solution (Dako S2023) was applied for 10 minutes at room temperature. After 2 washing steps with PBS, the slides were incubated with mouse monoclonal antibodies 6V12 or 6V32, which were elicited against HPyV-6 virus-like particles using previously reported methods.^{20 (link)} 6V12 (isotype IgG3)is specific for HPyV-6VP1, whereas 6V32 (isotype IgG1) cross-reacts with HPyV-7 VP1. Neither monoclonal antibody is reactive with the VP1 protein of MCPyV. After primary antibody binding, the slides were washed twice again with PBS and then incubated with biotinylated secondary antibody (K5003A, Dako) for 30 minutes at room temperature; after 2 more washing steps with PBS, streptavidin horseradish peroxidase (K5003B, Dako) was applied for 20 minutes at room temperature. Slides were washed twice in PBS and stained with ImmPACT NovaRED (Vector Laboratories) for 8 minutes at room temperature. After another wash in PBS, slides were counterstained with hematoxylin (Dako), rinsed in water, dehydrated, and mounted in Tissue-Tek Glas mounting medium (Sakura Finetek).