Betazoid dabkit

Sections were cut, deparaffinised, and rehydrated as described above. Antigen retrieval was performed for 30 s at 123°C in Antigen Unmasking Solution (Vector Laboratories, Burlingame, CA) using a pressure cooker followed by a cool-down period of 20 min. Reduction of peroxidases was accomplished by incubating for 8 min in Peroxidazed 1 (BioCare Medical, Concord, CA). Slides were sequentially blocked in Avidin-Biotin Blocking Kit (BioCare Medical), Background Sniper (BioCare Medical) and Rodent Block M (BioCare Medical) for 15 min, respectively. Biotinylated Hyaluronic Acid Binding Protein (Millipore, Billerica, MA) was added at a 1:800 dilution and incubated for 1 h at room temperature followed by a 10 min incubation with 4+ Streptavidin HRP Label (BioCare Medical). Staining was performed with Betazoid DABKit (BioCare Medical) for 5 min followed by counterstaining with CAT Hematoxylin (BioCare Medical) for 1 min.

3,3’-DAB staining was used to visualize the endogenous peroxidase activity of Hb.^30–32 (link) Four male WKY rats were used for DAB staining. A 45-min warm unilateral arterial clamp ischemia was performed followed by 2 h of reperfusion before harvesting the right (ischemic) and left (control) kidneys and placing in fixative solution. Paraffin-embedded sections of both control and IR tissue were cut at 5 µm and mounted on the same slide. Slides were then deparaffinized and hydrated to deionized water. Re-hydrated sections were then placed in a dark chamber and incubated with DAB solution (Biocare Medical Betazoid DAB Kit cat# BDB2004L) for 20 min, followed by rinsing in distilled water. Stained sections were counterstained with hematoxylin for 1 min, dehydrated in an ethanol series, and cleared in xylene before mounting. N = 3 slides each from rats undergoing either 45 min of either arterial or venous clamping without reperfusion or 45 min of venous clamping with saline perfusion were also stained for DAB.

Tissues were fixed in Z-Fix (Anatech LTD, Battle Creek, MI) and embedded in paraffin. Sections (5 µm) were dewaxed and rehydrated in deionized water. The endogenous peroxidase activity and non-specific background were blocked with 3% H₂O₂ (Fisher, Pittsburgh, PA) and Rodent Block M (Biocare Medical, Concord, CA), respectively. After an overnight incubation with a rat anti-mouse LAMP1 antibody (1∶500 dilution) at 4°C, sections were incubated with components from the Promark rat-on-mouse HRP-polymer kit (Biocare Medical); stain was developed with a Betazoid DAB kit (Biocare Medical).