Diaminobenzidine solution

Following deparaffinization and rehydration, tumor sections (3-μm thickness) were incubated in 0.3% H₂O₂ in methanol for 30 min at 37 °C to block endogenous peroxidase. The sections were then boiled in 10 mmol/L citrate buffer (pH 6.0) for 2 min in an autoclave. The anti-ASIC2 (Abcam) or anti-NFAT1 (Cell Signaling) antibody was added and the sections were incubated at 4 °C overnight. The sections were visualized by using the diaminobenzidine solution (DAKO, Carpinteria, CA, USA), and then lightly counterstained with hematoxylin. Sections without incubation with primary antibody served as negative controls. The intensity of staining (brown color) was semi-quantitatively scored as follows: 1, weak; 2, medium; 3, strong; and 4, very strong. The percentage of maximally stained tumor cells in each section was recorded (0, <5%; 1, 5–30%; 2, 30–50%; 3, >50%). High expression of ASIC2 was defined as a combined score for the intensity and area of staining that was larger than 4. The results were verified by two pathologists independently.

Histological staining of PFA-fixed spleen sections was performed as previously described^{46 (link)}. Murine kidneys were fixed in 4% PFA and embedded in paraffin for histological analysis. Sections (3 µm) were cut and mounted on aminopropyltriethoxysilane-coated slides, and H&E staining was performed to assess histological features. PAS staining was performed to evaluate mesangial expansion. For immunohistochemical staining, endogenous peroxidase was inactivated by treatment with 0.3% hydrogen peroxide in methanol for 30 min. Heat-induced antigen retrieval was performed by incubating sections with 10 mM Tris base containing 1 mM ethylenediaminetetraacetic acid (pH 9.0) in a pressure cooker for 8 min. Nonspecific binding sites were blocked with 0.25% casein solution (Dako, Glostrup, Denmark) for 5 min. Sections for C3 analysis were incubated for 1 h with a goat polyclonal anti-C3 antibody (1:200), followed by incubation with an anti-goat HRP polymer for 1 h. Sections for IgG staining were incubated with an anti-mouse HRP polymer for 30 min. The reaction products were developed with a diaminobenzidine solution (Dako). Imaging was performed by using BZ-9000 All-in-one Fluorescence Microscope (Keyence).

We prepared deparaffinized sections of untreated biopsy specimens of head and neck cancer for immunohistochemical staining for REG-I; they were initially autoclaved for 15 min at 121 °C, then were blocked with 0.3 % hydrogen peroxide in methanol for 30 min at room temperature and with 10 % BSA/TBS for 30 min at room temperature. All sections were kept overnight at 4 °C in phosphate-buffered saline containing anti-REG-I monoclonal antibodies (1:400 dilution, 2.5 μg/mL; BioVendor Laboratory Medicine, Inc., Evropska, Czech Republic), and were subsequently incubated for 20 min with Envision (Dako Corporation, Copenhagen, Denmark). The signal was detected by incubating the sections with diaminobenzidine solution (Dako) and hydrogen peroxide for one minute. We used image-J software as an objective method to measure the intensity of immunohistochemical staining for REG-I in biopsy specimens of head and neck cancer.