Super sensitive polymer hrp ihc

Formaldehyde-fixed tissues were transferred to a paraffin-embedded block, sectioned at 4-μm thicknesses. After tissue deparaffinization and rehydration, endogenous peroxidase activity was blocked by 10-min incubation at room temperature with absolute methanol containing 1% hydrogen peroxide. The tissue sections were incubated with a primary antibody against mouse anti-mortalin monoclonal antibody (C1-3), rabbit anti-p53 (sc-6243; Santa Cruz biotechnology, Santa Cruz, CA), and mouse anti-PCNA (M0879; DAKO, Carpinteria, CA) at 4 °C overnight. After incubation with the secondary antibody (Super Sensitive™ Polymer-HRP IHC, BioGenex) for 1 h at room temperature, the bound complexes were visualized by incubating tissue sections with 0.05% diaminobenzidine and 0.003% hydrogen peroxide. The sections were counterstained with Harris hematoxylin and then dehydrated and mounted. Mortalin, p53, and PCNA protein levels were semi-quantitatively analyzed using MetaMorph^® image analysis software (Universal Image Corp., Buckinghamshire, UK). Results are expressed as mean optical density of six different digital images per sample.

Formaldehyde-fixed tissues were transferred to a paraffin-embedded block, and 4 μm-thick sections were cut. After tissue deparaffination and rehydration, endogenous peroxidase activity was blocked by 10-min incubation at room temperature with absolute methanol containing 1% H₂O₂. The tissue sections were incubated with a primary antibody against rabbit anti-HMGB1 antibody (Abcam), rabbit anti-RAGE (Abcam), and monoclonal mouse anti-TLR4 (Imgenex, Midland, Canada) at 4 °C overnight. After incubation with secondary antibody (Super Sensitive™ Polymer-HRP IHC, Bio Genex) for 1 h at room temperature, the bound complexes were visualized by incubating the tissue sections with 0.05% diaminobenzidine and 0.003% H₂O₂. The sections were counterstained with Harris hematoxylin for nuclei, dehydrated, and mounted. The expression levels of HMGB1, TLR4, and RAGE were semi-quantitatively analyzed using MetaMorph® image analysis software (Molecular Devices, Sunnyvale, CA, USA). The results are expressed as the mean optical density (OD) for six different digital images per sample.