Flex trs high ph retrieval buffer

Immunohistochemistry for CXCR4 and CD99 was performed on formalin fixed paraffin embedded tumor sections using the Dako Autostainer Link (Dako, Carpinteria, CA). Following rehydration, sections were treated with heat induced epitope retrieval (HIER). For CXCR4 HIER was performed with FLEX TRS Low pH Retrieval buffer (pH 6.10) (Dako) for 20 minutes. For CD99 HIER was performed with FLEX TRS High pH Retrieval buffer (pH 9.01) (Dako) for 20 minutes. After peroxidase blocking, CXCR4 antibody (ab124824; abcam, Cambridge, MA) was applied at a dilution of 1:4000 at room temperature for 60 minutes, and CD99 antibody (M3601; Dako, Carpinteria, CA) was applied at a dilution of 1:100 at room temperature for 60 minutes. The FLEX HRP EnVision System (Dako) was used for detection. DAB chromagen was then applied for 10 minutes. Slides were counterstained with Harris Hematoxylin for 5 seconds and then dehydrated and coverslipped.