Hrp conjugated goat anti rabbit immunoglobulin igg h l

The skin and ankle joints were fixed in neutralized 10% formalin, embedded in paraffin, and sliced. For immunohistological analysis using AMC antibodies, we prepared modification buffer by mixing Reagent A (20% H₂SO₄, 25% H₃PO₄, and 0.025%FeCl₃) and Reagent B (1% diacetyl monoxime, 0.5% antipyrine, 1 M acetic acid) at a 2:1 ratio (volume/volume). The sections were covered with the modification buffer and incubated in a light-proof container at 37 °C for 2.5 h in order to modify citrulline residues. Then, the sections were incubated with rabbit AMC polyclonal antibodies diluted 1:3200 in 2% BSA in PBS to detect modified citrulline residues. Then, the sections were incubated with HRP-conjugated goat anti-rabbit IgG (H + L) (Bio-Rad, Hercules, CA, USA) for 30 min at room temperature. The sections were also stained with DAB (Nichirei Biosciences, Tokyo, Japan) and hematoxylin. To detect citrullinated histone 3 (CitH3), the sections were incubated with rabbit anti-CitH3 antibodies (Abcam, Cambridge, MA, USA) diluted 1:200 as primary antibodies. HRP-conjugated goat anti-rabbit immunoglobulin (IgG) (H + L) (Bio-Rad) diluted 1:1000 was used as a secondary antibody. The sections were also stained with DAB (Nichirei Biosciences) and hematoxylin.