Bond polymer refine hrp kit

Immunohistochemical IHC) staining was performed by Gavish Research Services on 7µm frozen sections using the Leica Bond max system (Leica Biosystems Newcastle Ltd, UK). Slides were dried at room temperature following pretreatment with epitope-retrieval solution (ER1, Leica Biosystems Newcastle Ltd, UK) followed by incubation with anti-Nudt2 ((#10484-1-AP, dilution 1:200, Proteintech, Biotest, Israel). Detection was performed using the Leica Bond Polymer Refine HRP kit (DS980 by Leica Biosystems Newcastle Ltd, UK). All slides were counter-stained with Hematoxylin and evaluated as a semi- quantitative analysis of the immunohistochemical reaction for NUDT2 was performed using a scoring scale as follows:

Mouse liver tissues were trimmed, fixed in 4% neutral buffered formalin, embedded in paraffin, and sectioned at 4 μm thickness. Slides were baked for 120 min at 60 °C, dewaxed, and pretreated with epitope-retrieval solution (ER, Leica Biosystems Newcastle Ltd., Newcastle Upon Tyne, UK), followed by incubation for 30 min with primary antibodies. Detection of CD3 (Abcam, Cambridge, UK, #Ab5690, 1:200 concentration) and Iba1 (Abcam, #Ab178847, 1:2000 concentration) was performed using the Leica Bond Polymer Refine HRP kit (Leica Biosystems Newcastle Ltd., Newcastle Upon Tyne, UK), while Ly6G (R&D Systems, Minneapolis, MN, USA, #MAB10371, 1:400 concentration) was detected using an anti-rat IgG secondary antibody (VC005 by R&D Systems, Minneapolis, MN, USA). All slides were counter-stained with hematoxylin. Hematoxylin and eosin (H&E) staining was imaged using an Olympus BX60 microscope equipped with a DP-73 camera at magnifications of x4, x10, x20, and x40. Grading for necrosis (by number of inflammatory cells per X20 High Power Field (HPF); 0 = none, 1 = 10–20 per HPF, 2 = 20–50 per HPF, 3 > 50 per HPF), fibrosis (by percentage of fibrosis of the tissue; 0 = 0%, 1 = <25%, 2 = 25–75%, 3 > 75%), and biliary hyperplasia (by number of extra ducts in portal area; 0 = 0, 1 = 2–10, 2 = 10–20, 3 > 20) were analyzed by Patho-Logica.

H460 tumors grown on CAM were collected at EDD18, washed in PBS, and fixed in 4% paraformaldehyde for 48 h at 4 °C. The tumors were then cleaned of any remaining CAM tissue, trimmed, and embedded in paraffin cassettes. IHC staining was performed on 4 µm FFPE sections using the Leica Bond max system (Leica Biosystems Newcastle Ltd., UK). Slides were baked for 30 min at 60 °C, dewaxed and pretreated with an epitope-retrieval solution: CD3 (Abcam, France), CD8 (Biorbyt, France) and CD4 (Abcam, France). Detection was performed using the Leica Bond Polymer Refine HRP kit (Leica Biosystems Newcastle Ltd., UK). All slides were counter-stained with Hematoxylin. Illustrative pictures were acquired with a Leica CTR 6500 confocal microscope (Leica Microsystems, Germany) at 20× magnification.