3 3 diaminobenzidine tetrahydrochloride dab

The paraffin-embedded tissues were sectioned continuously into 4-μm-thick sections. The tissue sections were dewaxed in xylene, rehydrated and rinsed in graded ethanol solutions. The antigens were retrieved by heating the tissue sections at 100 °C for 5 min in EDTA (1 mmol/L, pH 8.0) solution when necessary. The sections were then immersed in a 0.3% hydrogen peroxide solution for 10 min to block endogenous peroxidase activity, rinsed in phosphate buffered saline (PBS) for 5 min, and incubated with the WNT6 primary antibody (1:200 dilution, ab50030; Abcam, Cambridge, MA, USA) at 4 °C overnight. Subsequently, the slides were washed with 1× PBS and treated with a goat antibody against a mouse/rabbit secondary antibody (Envision; Dako, Glostrup, Denmark) at 37.5 °C for 30 min. Finally, the visualized staining was developed with 3,3'-diaminobenzidine tetrahydrochloride (DAB, Dako, Glostrup, Denmark), and all of the slides were counterstained with hematoxylin.

Formalin-fixed paraffin-embedded sections were evaluated by applying our optimized PTEN staining immunohistochemistry assay.33 (link) Sections were obtained at a thickness of 3 μm, dried for 1 h at 65°C, deparaffinized, rehydrated, and subjected to target retrieval in the pretreatment module PT Link (Dako) at 95°C for 20 min in Target Retrieval Solution (pH 9) (Dako). Endogenous peroxidase was blocked using peroxidase-blocking reagent (Dako), and this was followed by incubation with primary PTEN antibody (clone 6H2.1; Dako). The incubation time for the primary antibody was 40 min. EnVision FLEX+/HRP (Dako) (20 + 15 min) was used to amplify signal, and detection was done using 3,3′-diaminobenzidine tetrahydrochloride (DAB) as chromogen (Dako). Slides were counterstained with haematoxylin, dehydrated, and mounted. PTEN expression was nuclear and cytoplasmic. Appropriate negative controls were also tested: these involved omission of the primary antibody, and the inclusion of tissue samples known to show PTEN silencing. We used a histological staining score (Hscore) that takes into consideration the intensity of the staining, and the percentage of positive cells, by applying the formula: Hscore = (1 × % light staining) + (2 × % moderate staining) + (3 × % strong staining). This gives a score ranging from 0 (no immune reaction) to 300 (maximal immunoreactivity).

The joints collected for histology were fixed in 4% paraformaldehyde, decalcified in EDTA solution (0.25 mol/l, pH 7.4), and embedded in paraffin. Haematoxylin and eosin (H&E) and safranin-O/fast green staining were conducted with 6 μm-thick tissue sections. Immunohistochemistry was performed following a standard protocol. Briefly, tissue sections were regularly deparaffinized and rehydrated. The antigen was retrieved with citric acid buffer (pH 6.0) and microwave antigen retrieval for 15 minutes. After blocking with hydrogen peroxide and serum, the tissue sections were incubated with ANT3 antibodies (Abcepta) overnight at 4°C. HRP-labelled secondary antibody was incubated at 37°C for 60 minutes. The colour was developed by applying 3,3′-diaminobenzidine tetrahydrochloride (DAB) (DakoCytomation, USA), and the sections were counterstained with haematoxylin.^{24 (link)}