Endogenous peroxidase blocking

Each biopsy sample was fixed in 10% buffered formalin, dehydrated in graded alcohol series, and embedded in paraffin. Skin sections (thickness 3 μm) were deparaffinised, treated with endogenous peroxidase blocking (Dako, USA) and then with Dako Protein block (Dako, USA) to block non-specific binding. After blocking, sections were incubated with anti-IL-32 antibody (AbCam, UK). Visualisation of the primary antibodies was performed using EnVision Flex/HRP and DAB (diaminobenzidine) (both Dako, USA). No immunohistochemical staining was noted in negative control samples where the primary antibody was omitted. Sections were examined and photographed under light microscope (Olympus BX53). The number of positive cells was counted by two pathologists, blinded to tissue source and expressed as the mean of two observations for each sample. Results were reported as the median (range) of number of positive cells per microscopic field, considering the non-parametric distribution.

Heat treatment for 20 min was necessary before endogenous peroxidase blocking (DAKO, Glostrup, Denmark) for 5 min. Afterward, sections were incubated with a polyclonal antibody against ionized calcium binding adaptor molecule-1 (IBA-1, also known as allograft inflammatory factor-1 (AIF-1), 1:1000, overnight 4 °C; WAKO, USA).

As previously published [114 (link)], 98% formic acid immersion for 15 min, proteinase K (4 μg/mL; Roche, Reinach, Switzerland) treatment for 15 min at 37 °C and hydrated heating for 20 min preceded endogenous peroxidase blocking (DAKO, Glostrup, Denmark) for 5 min and incubation with the monoclonal antibody L42 (1:500, 30 min RT; DAKO, Glostrup, Denmark).

A pre-treatment consisting of hydrated heating at 121 °C in 10% citrate buffer for 20 min preceded the endogenous peroxidase blocking (DAKO, Glostrup, Denmark) for 5 min and incubation with different primary antibodies: polyclonal IL-1α (1:100, overnight 4 °C; ThermoFisher Scientific, Waltham, MA, USA), polyclonal IL-1RN (1:100, overnight 4 °C; Sigma, St. Louis, MO, USA), monoclonal 8H12 (1:20, overnight 4 °C; ThermoFisher Scientific, Waltham, MA, USA), or polyclonal IFNGR1 (1:1200, overnight 4 °C; ThermoFisher Scientific, Waltham, MA, USA) were used.

A pre-treatment consisting of hydrated heating at 121 °C in citrate buffer 10% for 20 min preceded the endogenous peroxidase blocking (DAKO, Glostrup, Denmark) for 5 min and incubation overnight 4 °C with different primary antibodies: polyclonal IL-1α (1:100; ThermoFisher Scientific, Waltham, MA, USA), polyclonal IL-1RN (1:100, Sigma, St. Louis, MO, USA), monoclonal 8H12 (1:40; ThermoFisher Scientific, Waltham, MA, USA) or polyclonal IFNGR1 (1:200, ThermoFisher Scientific, Waltham, MA, USA).

After endogenous peroxidase blocking (DAKO, Glostrup, Denmark) for 5 min, slides were incubated with a polyclonal antibody against glial fibrillary acidic protein (GFAP, 1:500, 30 min RT, DAKO, Glostrup, Denmark).

Synovia paraffin sections (thickness 3 μm) were deparaffinised, treated with endogenous peroxidase blocking (Dako, USA) and then with Dako Protein block (Dako, USA) to block non-specific binding. After blocking, sections were incubated respectively with anti-IL-1 (sc-7884, 1:100, Santa Cruz Biotechnology, USA), anti-IL-6 (sc-130326, 1:100, Santa Cruz Biotechnology, USA), anti-TNFα (sc-8301, Santa Cruz Biotechnology, USA) and anti-FeH antibody (sc-376594, 1:200, Santa Cruz Biotechnology, USA). Visualisation of the primary antibodies was performed using EnVision Flex/HRP and DAB (diaminobenzidine) (both Dako, USA). No immunohistochemical staining was noted in negative control samples where the primary antibody was omitted. Sections were examined and photographed under light microscope (Olympus BX53).