Peroxidase goat anti rabbit

After blocking of endogen peroxidase by 0.5% H₂O₂ in PBS for 10 min at room temperature and repeated washings in PBS, samples were pre-incubated with a blocking 179 buffer (0.1% BSA in PBS) for 60 min at room temperature, and then incubated in goat anti-collagen-Type I, (1:400, SouthernBiotech, Birmingham, AL, USA), or rabbit anti-collagen-III, N-terminal antibody (1:100, Abcam, Cambridge, UK), overnight at 4 °C. After repeated PBS washings, samples were maintained for 1 h in secondary antibody (Collagen Type I), peroxidase Rabbit Anti-Goat and (Collagen III) peroxidase-Goat Anti-Rabbit (1:300, Jackson Immu-noResearch Laboratories, Inc., West Grove, PA, USA), and then washed in PBS. The reaction was developed with 3,3′-diaminobenzidine (Liquid DAB plus substrate Chromogen System kit; Dako, Glostrup, Denmark). Negative controls were conducted by the omission of the primary antibody, confirming the specificity of the immunostaining.

After blocking of endogen peroxidase by 0.5% H₂O₂ in PBS for 10 min at room temperature and repeated washings in PBS, samples were pre-incubated with a blocking buffer (0.1% BSA in PBS) for 60 min at room temperature and then incubated in goat anti-collagen type I, (1:400, SouthernBiotech, Birmingham, AL, USA), or rabbit anti-collagen-III, N-terminal antibody (1:100, Abcam, Cambridge, UK), overnight at 4 °C. After repeated PBS washings, samples were maintained for 1 h in secondary antibody (collagen type I), peroxidase rabbit anti-goat and (collagen III) peroxidase goat anti-rabbit (1:300, Jackson ImmunoResearch Laboratories, Inc.), and then washed in PBS. The reaction was developed with 3,3′–diaminobenzidine (Liquid DAB plus substrate Chromogen System kit; Dako). Negative controls were conducted via omission of the primary antibody, confirming the specificity of the immunostaining (Appendix AFigure A1B,C).