Harry s hematoxylin

Immunocytochemistry analysis was performed on coverslip-adherent cells by the labeled streptavidin biotin method using a LSAB kit (DAKO Japan, Kyoto, Japan). The samples were incubated with 0.1% Triton X-100 solution for 1 h, washed 3 times in PBS and treated for 40 min at room temperature with 10% BSA. In addition, coverslips were incubated overnight at 4 °C with the primary antibodies (anti-p53, anti-p65-NF-kB, anti-TGF-β1, diluted 1:400 and anti-Nrf2, diluted 1:300, Santa Cruz Biotechnology). Next, secondary antibody treatment was performed (2 h at room temperature), and horseradish peroxidase activity was visualized by treatment with H₂O₂ and 3,3′-diaminobenzidine (DAB) for 5 min. For the final step, the sections were weakly counterstained with Harry’s hematoxylin (Merck). For each case, negative controls were performed by omitting the primary antibody. Intensity and localization of immunoreactivities against the primary antibody used were examined on all coverslips using a photomicroscope (Olympus BX41, Olympus Optical Co., Ltd., Tokyo, Japan). For image analysis, color images of representative areas (400×) were digitally captured. For p53, p65-NF-kB and Nrf2 analyses, the percentage of cells with labeled nuclei were calculated.

Nitrotyrosine (NT) is a residue formed by the action of peroxynitrite (derived from the reaction of nitric oxide and superoxide anion) on proteins. Paraffin-embedded sections were heated (30 min, 65°C), deparaffinized, and rehydrated. Sections were treated at room temperature with 2% bovine serum albumin and incubated overnight at 4°C with primary mouse anti-human antibodies against NT-labeled residues (diluted 1 : 300, Santa Cruz Biotechnology, clone sc-32757, USA), previously validated for human samples [13 (link)]. The secondary antibody, horseradish peroxidase, and 3,3′-diaminobenzidine (DAB) were provided by the commercial kit (Dako LSAB, Germany). In the last step, sections were weakly counterstained with Harry's hematoxylin (Merck). For each case, negative controls were performed on serial sections by omitting the primary antibody incubation step. The intensity and localization of the immunoreactivity were examined with a photomicroscope (Leica DM 2500 and Leica DFC280, Leica, Germany).

ACL tissue samples (length: ~2 mm) were fixed overnight in 4% PFA solution, dehydrated and finally embedded in paraffin. After slicing, the 7 µm sections were rehydrated in a descending alcohol series and prepared for hematoxylin eosin (HE) and alcian blue (AB) staining.
For HE staining paraffin sections and glass cover slips were incubated for 6 min in Harry’s hematoxylin (Sigma-Aldrich, Munich, Germany), before rinsed in tap water and counterstained for 4 min in eosin (Carl Roth GmbH, Karlsruhe, Germany).
For AB performance, the tissue sections were rehydrated in a descending alcohol series and subsequently incubated for 3 min in 1% acetic acid before incubated for 30 min in 1% AB staining solution (Carl Roth GmbH, Karlsruhe, Germany). After rinsing in 3% acetic acid and a 2 min washing step in A. dest., ligamentocyte cell nuclei were counterstained for 5 min in nuclear fast red aluminium sulphate solution (Carl Roth GmbH).
HE and AB stained sections were covered with Entellan (Merck-Millipore, Darmstadt, Germany). Photos were taken using a DM1000 LED light microscope (Leica, Wetzlar, Germany).