Dako lsab 2 system hrp

We examined the localization of SCX in three IPF and three healthy lung tissues. Immunohistochemical analyses were performed as described [66 (link)]. Briefly, paraffin-embedded lung tissues were obtained from biopsy or autopsy specimens of individuals with IPF or HP, and controls, in compliance with institutional review board-approved protocols. Three millimeter lung sections were dewaxed and rehydrated, and peroxidase activity was blocked with 3% H₂O₂ in methanol for 30 min, followed by a citrate buffer treatment to retrieve the antigen. The lung sections were incubated overnight at 4 °C with an anti-human SCX rabbit polyclonal antibody (1:25; Antibodies online ABIN9670006). A secondary biotinylated anti-immunoglobulin, followed by horseradish peroxidase-conjugated streptavidin (DAKO LSAB^®2 System-HRP, Dako North America, Inc. K0609, Carpinteria, CA, USA) and the 3-amino-9-ethyl-carbazole (BioGenex, HK129-5K, San Ramon, CA, USA) substrate were used following the manufacturer’s instructions. Pictures of the hematoxylin-counterstained slides were acquired with a Nikon microscope equipped with the NIS-Elements AR software.

After deparaffinization, revitalization, and rehydration, the tissue slides were treated with a 3% H₂O₂ solution for 10 min for blocking endogenous peroxidases. Washing with Tris buffer was used after each handling step. The sections were incubated with the primary antibody rabbit anti-caspase 3 (1:500; Bioss, Woburn, MA, USA) for 30 min at room temperature. The specimen was then incubated by sequential 10 min. incubation with biotinylated anti-rabbit secondary antibody and peroxidase-labeled streptavidin conjugated to HRP (DAKO LSAB^®2 System-HRP; Dako, Denmark). The colour of the sections was developed after incubation with the DAB-chromogen solution (Dako). The sections were then counterstained with Mayer’s hematoxylin and mounted with Entellan (Merck, Kenilworth, NJ, USA). The slides were viewed with an Olympus BX41 microscope (Olympus, Japan). The image capture was performed with Quick Photo Micro software, version 2.2 (Olympus). The density of activated caspase-3 immunoreactive cells (dark-brown cytoplasm; caspase 3-IR) was measured randomly from three sites within each section and was calculated as the total number of caspase 3-IR cells in the field.

Specific primary antibody against CYP2U1 (ab252975, Abcam, Cambridge, MA, USA) was used for IHC at a dilution of 1:50. Immunohistochemical staining was performed as per instructions of the manufacturer. Sections of the TMA were dewaxed and rehydrated through a chain of xylenes and alcohols. Following that, the sections were incubated in 3% H₂O₂ diluted in methanol for 30 min in order to close biologically active sites of endogenous peroxidases. Antigen retrieval was implemented with citrate buffer (pH 8.0) at 98°C for 20 min. The slides were then allowed to cool to room temperature. After that, slides were incubated with primary antibody for 60 min at room temperature, and then incubated with biotinylated secondary antibody employing the Dako LSAB2 System-HRP (K0672, Dako Cytomation, Carpinteria, CA, USA) for 30 min, followed by washing with buffer so as to remove uncombined antibody. 3,3′diaminobenzidine (DAB) was utilized as the chromogen away from light applied for 3 min. Finally, sections were further washed within water and slightly counterstained with hematoxylin for 2 min. Slides with PBS, superseding the specific primary antibody, acted as blank controls.