Immunohistochemistry was performed for Mac-2 (total macrophages) and CD31 (endothelial cells) to test the retention of immunoreactivity in sections following OPT processing and imaging. In brief, rehydrated paraffin sections were incubated with primary rat anti-mouse/human Mac-2 antibodies (1:4,000 dilution overnight; Cedarlane, Ontario, Canada) and rabbit anti-mouse CD31 antibodies (1:50 dilution overnight; Abcam, UK) before incubation with biotinylated rabbit anti-rat IgG (1:200 dilution 40 min; Vector Labs, UK) or biotinylated goat anti-rabbit IgG secondary antibodies (1:200 dilution 40 min; Vector Labs), respectively. The sections were then treated with avidin and biotinylated horseradish peroxidase macromolecular complex (3 drops/slide 30 min; Vector Labs) before coloring by 3,3′-diaminobenzidine substrate kit (100 μl/slide 3–5 min; Vector Labs).
Biotinylated horseradish peroxidase macromolecular complex
Manufactured by Vector Laboratories
Sourced in United States
Biotinylated horseradish peroxidase macromolecular complex is a conjugate composed of the enzyme horseradish peroxidase (HRP) and biotin. This macromolecular complex is used as a detection reagent in various biological assays and techniques.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti mouse cd31 antibody, by Abcam (1 mentions)
Biotinylated rabbit anti rat igg, by Vector Laboratories (1 mentions)
Biotinylated goat anti rabbit igg secondary antibody, by Vector Laboratories (1 mentions)
3 3 diaminobenzidine substrate kit, by Vector Laboratories (1 mentions)
Anti claudin 4, by Abcam (1 mentions)
3 amino 9 ethylcarbazole, by Vector Laboratories (1 mentions)
Mouse anti lc3 monoclonal antibody, by Santa Cruz Biotechnology (1 mentions)
Mouse anti beclin1 monoclonal antibody, by Abcam (1 mentions)
Biotin conjugated anti mouse igg, by Vector Laboratories (1 mentions)
3 protocols using biotinylated horseradish peroxidase macromolecular complex
1
Immunohistochemical Evaluation of Macrophages and Endothelial Cells
2
Immunohistochemical Analysis of Claudin-4 in Mouse Lungs
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Mouse lung sections were made as previously described,25 (link) and then treated for non-specific binding with 1.5% goat serum and incubated with the anti-claudin-4 (1:100, Abcam). The next day, sections were incubated with avidin and biotinylated horseradish peroxidase macromolecular complex (Vector Laboratories, Burlingame, CA, USA). Color reaction was developed by staining with a liquid DAB + substrate kit (Golden Bridge International Inc., Mukilteo, WA, USA). After immnohistochemical staining, the slides were counterstaining with Herris's hematoxylin for 1 minute. Images were analyzed with the Image J program (National Institutes of Health, Bethesda, MD, USA), and stain density was quantified with an average of claudin-4 arbitrary density numbers from 6–8 fields.
3
Immunohistochemical Analysis of Autophagy and Oxidative Stress
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Myocardial tissue samples from the remote non-infarcted region were fixed in 10% neutral-buffered formalin, embedded with paraffin, and then sectioned. The paraffin sections, with a thickness of 4 μm, were deparaffinated and rehydrated with xylene and graded alcohol series. The tissue sections were washed in tap water and then incubated with 0.3% H 2 O 2 in methanol for 20 min at room temperature. The tissue sections were washed in phosphate-buffered saline (PBS), blocked with 5% goat serum in PBS, incubated with a mouse anti-LC3 monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-Beclin1 monoclonal antibody (Abcam, Cambridge, MA, USA) or mouse anti-4HNE monoclonal antibody (EDM Millipore, Billerica, MA, USA), and then incubated with biotin-conjugated anti-mouse IgG (Vector Laboratory, Burlingame, CA, USA). The sections were incubated with avidin and biotinylated horseradish peroxidase macromolecular complex (Vector Laboratory), and stained with 3-amino-9-ethylcarbazole (Vector Laboratory) and hematoxylin. For negative control, the primary antibody was omitted. The samples were examined under a light microscope.
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