Rabbit anti mouse cd45

Tissues were fixed in 10% neutral buffered formalin, processed and embedded in paraffin, and sectioned at 4 μm thickness using standard histologic procedures. Slides were dewaxed using xylene and rehydrated with a graded series of ethanol using a DAKO Coverstainer (DAKO, Agilent Technologies). Antigen retrieval was performed in a high pH buffer using PT Link (DAKO, Agilent Technologies) at 95°C for 20 minutes. IHC was carried out using a DAKO Autostainer Link 48 platform (DAKO, Agilent Technologies). Briefly, slides were blocked for endogenous peroxidases and subsequently stained using the following primary antibodies: rabbit anti-mouse CD8 (D4W27 at 1:200), Rabbit anti-mouse CD45 (Cell Signaling Technology D3F8Q at 1:200), and Rabbit anti-mouse CD31 antibodies (Abcam EPR17259 at 1:500) in Da Vinci diluent (Biocare Medical). MACH2 Rabbit AP polymer (Biocare Medical) was used to detect primary antibodies, followed by detection using Enzo Red chromogen (Enzo Life Sciences). Slides were then counterstained with Tacha's Hematoxylin (Biocare Medical), dehydrated, and coverslipped using the DAKO Coverstainer. Once dried, slides were scanned using a 3D Histech Pannoramic MIDI II Scanner (3DHISTECH), then image/spatial analyses were performed using HALO software (Indica Labs).