Mouse hrp polymer kits

Renal allograft tissues containing full cross-sections were immediately fixed in acid methanol (60% methanol and 10% acetic acid. Paraffin-embedded sections (5µm) were subjected to high-temperature antigen retrieval and paraffin removal in Trilogy (Cell Marque, Hot Springs, AR) in a pressure cooker. Endogenous peroxidase activity was blocked by incubation with 0.3% H₂O₂, and nonspecific protein interactions were blocked by incubation with a serum free protein block (DAKO, Carpinteria, CA). Slides were incubated with one of the following primary antibodies: monoclonal rat antibody to mouse Mac2, a marker of inflammatory macrophages (Cedarlane Laboratories, Burlington, NC), rat anti-human/mouse CD3 mAb (AbD Serotec, Raleigh, NC) or rabbit polyclonal antiserum to mouse C4d for 60 minutes at room temperature. Staining antibodies were visualized using rat or goat on mouse HRP-Polymer Kits (Biocare Medical, Concord, CA) followed by diaminobenzidine and counterstained with hematoxylin. Images were captured with a 12-megapixel DS-Ri1 Digital Camera using NIS-Elements software (Nikon, Melville, NY).

Full cross sections of allografts were immediately fixed in acid methanol (60% methanol and 10% acetic acid). Paraffin-embedded sections (5 mm) were subjected to high temperature antigen retrieval and paraffin removal in Trilogy (Cell Marque, Hot Springs, AR) in a pressure cooker. Endogenous peroxidase activity was blocked by incubation with 0.3% H2O2, and nonspecific protein interactions were blocked by incubation with a serum-free protein block (DAKO, Carpinteria, CA). The following primary antibodies were used to detect CD4 (ab183685), CD8 (ab217344), LCN2 (ab70287), PD-1 (ab214421) and Ki-67 (ab16667) from Abcam (Cambridge, MA), granzyme B (AF1865) and PD-L1 (rabbit MAB 90781; clone 2096C from R&D Systems, Minneapolis, MN). The primary antibody to mouse C4d was a previously described and validated polyclonal rabbit antibody ^{28 (link)}. Primary antibodies were visualized using rat, rabbit, and goat on mouse HRP-Polymer Kits (Biocare Medical, Concord, CA) as secondary antibodies followed by diaminobenzidine (DAB). For double stains Vina green, Vulcan Fast Red (Biocare Medical, Concord, CA) or Vector Blue (Vector Laboratories, Burlingame, CA) chromogens were used. Sections were counterstained with hematoxylin or Periodic acid-Schiff (PAS) from Richard-Allan Scientific (Kalamazoo, MI).

Kidney grafts were harvested and fixed in acid methanol (60% methanol and 10% acetic acid). Paraffin-embedded sections (5μm) were subjected to high temperature antigen retrieval and paraffin removal in Trilogy (Cell Marque, Hot Springs, AR) in a pressure cooker. Endogenous peroxidase activity was eliminated by incubation with 0.03% H₂O₂ for 10 min. Nonspecific protein interactions were inhibited by incubation with serum-free protein block (DAKO, Carpinteria, CA). The slides were then stained using Masson’s Trichrome Stain Kit (American MasterTech, Lodi, CA) and with the following primary antibodies: monoclonal rat antibody to mouse Mac2 (Cedarlane Laboratories, Burlington, NC), rabbit polyclonal anti-CD3 antibody (Abcam, Cambridge, MA), and rabbit polyclonal antiserum to mouse C4d produced as described previously (43 (link)). Staining antibodies were visualized using rat or rabbit on mouse HRP-Polymer Kits (Biocare Medical, Concord, CA) as secondary antibodies followed by DAB and counterstained with hematoxylin. Slides were viewed under light microscopy, and images captured using ImagePro Plus (Media Cybernetics, Silver Springs, MD).