We prepared and stained formalin-fixed, paraffin-embedded 5-micron-thick lung sections using standard procedures. Briefly, sections were deparaffinized through graded alcohols and washed in PBS. Heat-activated antigen retrieval was performed in a microwave oven using a citrate buffer (Target Retrieval Solution, S1699, DAKO) for 10 minutes. Endogenous peroxidases were blocked using a peroxidase blocker (DAKO Cytomation), and the slides were blocked for nonspecific protein binding by incubating in 10% normal serum for 45 minutes. Tissues were immunostained with a rabbit primary IgG to Irp2 (LS-B48, LS Bio) at a 1:50 dilution or with mouse anti-cytochrome C, (556433, BD Biosciences) at a 1:250 dilution or with rabbit monoclonal IgG to cytochrome C EPR1327 (D00355 Abcam) at a 1:50 dilution, or with rabbit anti-LC3B (L7543, Sigma Aldrich) at a 1:400 dilution and signals were developed using the VECTASTAIN Elite ABC Kit (PK-6101; Vector Laboratories), according to the manufacturers protocol. Hematoxylin and Eosin stains were purchased from Sigma Aldrich and staining was carried out as per manufacturer’s instructions. The negative control consisted of substituting PBS for the primary antibody.
Peroxidase blocker
Manufactured by Agilent Technologies
Peroxidase blocker is a reagent used in immunohistochemistry and other laboratory techniques to inhibit endogenous peroxidase activity in tissue samples. It helps prevent non-specific staining and background signal, allowing for more accurate detection of target analytes.
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2 protocols using peroxidase blocker
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Immunohistochemical Analysis of Lung Tissue
2
Immunohistochemical Analysis of Lung Tissue
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We prepared and stained formalin-fixed, paraffin-embedded 5-micron-thick lung sections using standard procedures. Briefly, sections were deparaffinized through graded alcohols and washed in PBS. Heat-activated antigen retrieval was performed in a microwave oven using a citrate buffer (Target Retrieval Solution, S1699, DAKO) for 10 minutes. Endogenous peroxidases were blocked using a peroxidase blocker (DAKO Cytomation), and the slides were blocked for nonspecific protein binding by incubating in 10% normal serum for 45 minutes. Tissues were immunostained with a rabbit primary IgG to Irp2 (LS-B48, LS Bio) at a 1:50 dilution or with mouse anti-cytochrome C, (556433, BD Biosciences) at a 1:250 dilution or with rabbit monoclonal IgG to cytochrome C EPR1327 (D00355 Abcam) at a 1:50 dilution, or with rabbit anti-LC3B (L7543, Sigma Aldrich) at a 1:400 dilution and signals were developed using the VECTASTAIN Elite ABC Kit (PK-6101; Vector Laboratories), according to the manufacturers protocol. Hematoxylin and Eosin stains were purchased from Sigma Aldrich and staining was carried out as per manufacturer’s instructions. The negative control consisted of substituting PBS for the primary antibody.
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