Envision flex dab kit

All the histopathological sections were stained on the Dako Omnis platform with Envision Flex Dab kit. Antibodies to vimentin (GA63061), E-cadherin (GA05961), Ki-67 (GA62661), beta-catenin (IR70261), cytokeratin 7 (A61961) and CD11b (WT.5) were added to individual sections. Slides were dehydrated and mounted with cover slips. Positive cells were identified visually. In the case of Ki-67, the percentage of positive cells was calculated by dividing the number of positive cells by the total number of cells in each high-power field (40× magnification). For CD11b infiltration, the number of positive stained cells per high power field (HPF) was determined. A total of 10 random fields was analyzed per sample from at least 3 specimens. For the remaining IHC stains, the immunoreactivity scoring system (IRS) was used as proposed by Remmele and Stegner (REF) with slight modification as follows: IRS = the product of staining intensity (SI) and percent positive staining (PP). SI was quantified as 0, negative; 1, weak; 2, moderate; and 3, strong. PP was determined as 0, negative; 1, 1–10% positive cells; 2, 11–50% positive cells; 3, 51–80% positive cells; 4, 81–100% positive cells. An IRS score of greater or equal to 4 was considered as a positive staining result [34 (link)]. A total of 10 fields were analyzed.

An immunohistochemistry assay was utilized to detect the PARP1 levels in the tissue microarray sections. Briefly, 4 μm slides were treated with 3% hydrogen peroxide-containing methanol, followed by incubation with 10% goat serum for blocking. After washing, the slides were incubated with anti-PARP1 antibody (1:2000, ab227244, Abcam, Cambridge, UK) and then EnVision™ FLEX/HRP (Dako, Glostrup, Denmark). Subsequently, EnVision™ FLEX DAB kit (Dako, Glostrup, Denmark) and hematoxylin were applied for staining. The PARP1 expression was analyzed based on the number of positive cells per mm2 by the imaging system (Vectra Polaris, PerkinElmer, Waltham, MA). The median of the positive cells per mm2 was considered as an watershed of positive or negative individuals.