3 amino 9 ethylcarbazole aec substrate chromogen

Lungs were fixed in 4% formaldehyde, paraffin embedded, sliced into 3-μm sections, deparaffinized, and antigen unmasked in citrate buffer solution (0.01 M pH 6.0). Then, endogenous peroxidase activity was blocked for 10 min at room temperature with 0.3% H₂O₂. Slides were incubated with rabbit primary anti-tryptase and anti-PAR-2 antibodies (Santa Cruz Biotechnology) in PBS for 1 h and with biotinylated anti-rabbit IgG secondary antibodies for 10 min at room temperature, and detected by the peroxidase-conjugated avidin-biotin complex reaction with 3-amino-9-ethylcarbazole AEC+/substrate chromogen (Dako, Hamburg, Germany). Sections were then counterstained with Mayer’s hematoxylin (Dako). Images were obtained using an Olympus (BX50) microscope (Olympus, Hamburg, Germany) and an Olympus digital camera.

Following routine paraffin embedding, 5 μ m microtome tissue sections were placed on positively charged glass slides. Tissues were deparaffinized, rehydrated with graded alcohols, and treated with 88% formic acid digestion prior to heat-induced epitope antigen retrieval in the 2100-Retriever^TM (Prestige Medical). Following retrieval, endogenous peroxidase activity was quenched prior to prion epitope detection with anti-prion antibody BAR224 (Caymen chemical) at 2 μ g/mL concentration and Envision+^TM anti-mouse HRP-labeled polymer (Dako). Following antibody treatment, antigen was visualized with 3-amino-9-ethylcarbazole (AEC) substrate chromogen (Dako). Tissue sections were counterstained with Meyer’s hematoxylin (Dako) and 0.1% bicarbonate bluing reagent prior to coverslipping with aqueous mounting media (Dako).