Ab2020

Human paraffin embedded tissue microarrays of DLBCL were purchased from US Biomax (LY1002 and LY1001b; US Biomax, Rockville, MD, USA). The slide was deparaffinized with xylene for 5 min, followed by two changes of xylene; the slides were then rehydrated. The slides were incubated with 3% H₂O₂ for 10 min to block peroxidase activity and subsequently incubated with blocking solution (2% FBS and 1% BSA) for 1 h. The primary antibody against SHP-1 (ab2020; Abcam, Cambridge, MA, USA) was used at 1:100 dilution for 1 h incubation at room temperature. The slides were washed three times with PBS and detected using the EnVision Detection Systems Peroxidase/DAB, Rabbit/Mouse kit (Agilent, Santa Clara, CA, USA). The slides were counterstained with hematoxylin and subsequently dehydrated and mounted. The IHC intensity of SHP-1 was determined independently by a qualified pathologist, and rated as a scale of 0 to 3 +, and an H-score of 0–300 based on percentage of cells stained at different intensities were assigned to each sample. Median value of positive IHC H-score was used as a cut-off at 41.25 for defining high versus low expression of SHP-1.