Biotinylated anti goat igg

HE staining and immunohistochemical staining was performed as previously described [26 (link)]. Briefly, for immunohistochemical staining, paraffin-embedded tissues, sectioned at 4 µm thickness, were dewaxed and boiled in Tris-EDTA buffer (10 mM Tris Base, 1mM EDTA-2Na, 0.05% Tween 20, pH 9.0) for 20 minutes. After blocking, anti-MRP8 (Santa Cruz Biotechnology, Santa Cruz, CA), anti-MRP14 (Santa Cruz Biotechnology), anti-CD3 antibody (Santa Cruz Biotechnology) or anti-CD45R (BD Biosciences, San Jose, CA) was applied to the serial sections of tissues. After washing with PBS, sections were incubated with biotinylated anti-goat IgG (Nichirei Bioscience, Tokyo, Japan) or biotinylated anti-rat IgG (Nichirei Bioscience), followed by incubation with alkaline phosphatase-conjugated streptavidin (Nichirei Bioscience). Finally, enzymatic color development was performed by using 4-[(4-amino-m-tolyl) (4-imino-3-methylcyclohexa-2,5-dien-1-ylidene)methyl]-o-toluidine monohydrochloride (new fuchsine, Nichirei Bioscience). For quantitative analyses of infiltrating MRP14⁺ and MRP8⁺ cells in the tissues, the number of MRP14⁺ or MRP8⁺ cells in the immunohistochemically stained tissues was counted in 5 random microscopic fields at 400x magnification.

Immunohistochemical staining was performed as previously described [24 (link)]. Briefly, for immunohistochemical staining, paraffin-embedded tissues, sectioned at 4 μm thickness, were dewaxed and boiled in Tris-EDTA buffer (10 mM Tris Base, 1 mM EDTA-2Na, 0.05% Tween 20, pH 9.0) for 20 minutes. After blocking, anti-MRP8 and anti-MRP14 antibody (Santa Cruz Biotechnology, Texas, USA) was applied to the serial sections of tissues. After washing with PBS, sections were incubated with biotinylated anti-goat IgG (Nichirei Bioscience, Tokyo, Japan). Finally, enzymatic color development was performed by using 4-[(4-amino-m-tolyl) (4-imino-3-methylcyclohexa-2, 5-dien-1-ylidene) methyl]-o-toluidine monohydrochloride (new fuchsine, Nichirei Bioscience).
The number of MRP8⁺ cells in the immunohistochemically stained spleen were counted in 10 random microscopic fields at 200× magnification. For the spleen, the cells in the red pulp were counted.