Bond 3 ihc autostainer

Immunohistochemical (IHC) staining from human tissue microarray or mouse xenograft samples was performed on 3-μm-thick sections from formalin-fixed paraffin-embedded samples and dried in a 37 °C oven overnight. Hematoxylin/eosin (H&E) staining was performed with H&E Leica Kit Infinity for a full automated autostainer (Leica ST5020). IHC staining for Ki67 and MGP antibodies was performed using Bond III IHC autostainer for full Automated Immunohistochemistry (Leica biosystems). Antigen was unmasked with Tris-EDTA pH 9 (Bond Epitop Retrival Solution 2 Leica, cat# AR9640). Both mouse monoclonal anti-Ki67 CloneMIB-1 (Dako, cat# M7240) and rabbit polyclonal antibody MGP (ProteinTech, cat# 10734-1-AP) were used at 1:200. The antibodies were diluted with Bond Primary Antibody Diluent AR9352 Leica. BOND IHC Polymer Detection Kit (cat# DS9800) was used to stain both antibody with DAB Cromogen. IHC samples were counterstained using Hematoxylin solution (Leica, cat# RE7107-CE). Pictures of stained sections were acquired with the Aperio ScanScope XT instrument. Ki67 and MGP staining were analyzed and scored by a board-certified pathologist (GB).

Blood smears were stained with May-Grünwald-Giemsa (Sigma Aldrich) according to the Sigma-Aldric Giemsa stain protocol (Procedure No. GS-10). Femor was first fixed overnight in 4% formaldehyde, followed by decalcification using osteodoc (Bio-optica, Cat# 05-M03005) following the manufacturer’s instructions. Spleen was fixed overnight in 4% formaldehyde. Fixed tissues were embedded in the parafilm with LogosJ Processor (Milestone). Sections of 3 micron were cut and left for an overnight incubation at 37 °C before staining. IHC staining for Ki67-SP6 and cleaved caspase3 antibodies was performed using Bond III IHC auto-stainer for full Automated Immunohistochemistry (Leica biosystems). For both antibodies antigen was unmasked with Tris-EDTA pH 9 (Bond Epitop Retrival Solution 2 Leica AR9640) followed by peroxidase blocking using Bond Polymer Refine Detection Kit (DC9800). Tissues were washed and then incubated with primary antibodies diluted in Bond Primary Antibody Diluent AR9352 Leica. Subsequently, tissues were incubated with polymer for 10 min and developed DAB Chromogen following by Hematoxylin as counterstain. Pictures of stained sections were acquired with the HistoFluo microscope (Leica DM6). Primary antibodies used: anti-Ki67monoclonal antibody (SP6) (Invitrogen, #MA5-14520, 1:200), anti-cleaved caspase3, monoclonal antibody (Asp175) (Cell Signaling, #9661; 1:200).