Aminoethylcarbazole aec kit

After deparaffinization, rehydration, blocking of endogenous peroxidase, antigen retrieval, blockade of nonspecific protein binding (0.4% bovine serum albumin; Sigma‐Aldrich Co), the specimens were incubated with the primary antibodies directed to laminin and fibronectin (Abcam), followed by incubation with anti‐rabbit secondary antibody (Abcam) and streptavidin‐horseradish peroxidase. Aminoethylcarbazole (AEC kit; Invitrogen) was used as the substrate–chromogen system and the slides were counterstained with Mayer's hematoxylin (Sigma‐Aldrich Co). The slides were examined under a light microscope (Nikon). Laminin and fibronectin deposits were quantified using the ImageJ software (NIH) and we analyzed at least 10 pictures/microscopy field. The results were expressed as area fraction (representative of the percentage of the stained area).

Immunohistochemistry was based on the method reported by Morgado and collaborators[20 (link)]. Briefly, spleen fragments frozen in OCT resin (Sakura) were cut into 3–5 μm-thick sections and mounted onto microscope slides (silanized slides; DakoCytomation, Carpinteria, CA, USA). The slides were fixed in acetone PA (Merck, Darmstadt, Germany) and subjected to hydration, endogenous peroxidase blockage (peroxidase blocking reagent; Dako) and nonspecific staining blockage (0.4% BSA; Sigma, USA). The specimens were then incubated with primary antibodies directed against CD3⁺, CD4⁺, CD8⁺, CD21⁺, IFN-γ⁺, IL-10⁺ (Serotec), Ki-67⁺ (eBioscience), laminin, fibronectin, ADAM-10 (Abcam) or MMP-9 (Serotec), followed by incubation with the Labelled Polymer (Envision System-HRP, Dako). Aminoethyl carbazole (AEC kit; Invitrogen) was used as the substrate-chromogen system, and the slides were counterstained with Mayer’s hematoxylin (Sigma). The slides were examined under a light microscope (Zeiss), and the number of marked cells/mm² tissue in the red pulp was determined. Two blinded readers evaluated the slides. Laminin and fibronectin deposits were quantified using ImageJ 1.48v software (NIH, USA), and the results are presented as the percentage of the marked area. Primary antibody suppression was used as the negative control reaction.