Immunohistochemical Detection of Histone Modifications

Immunohistochemistry was performed with the indirect enzyme-labelled antibody method, as described previously [1 (link), 17 (link), 27 (link)]. For the detection of histone H3, H3K4me3, H3K9ac, H3K18, H3K23ac, H3K27me3, and H3S10phos, paraffin sections of mouse testis were deparaffinized with toluene and dehydrated in serially graded ethanol solutions. The sections were autoclaved in a 10 mM citrate buffer (pH 6.0) for 15 min at 120°C. Then, the sections were preincubated with 500 µg/ml normal goat IgG dissolved in 1% BSA in PBS, pH 7.4, for 1 hr. Unless otherwise specified, all reactions were conducted at room temperature. Then the sections were reacted 16 hr with first antibodies in 1% BSA in PBS. After incubation, the sections were washed with 0.075% Brij 35 in PBS three times for 15 min each. The sections were then incubated with HRP-conjugated goat anti-rabbit IgG in 1% BSA in PBS for 1 hr. After washing with 0.075% Brij 35 in PBS three times for 15 min each, the sites of HRP were visualized with DAB and H₂O₂ in the presence of nickel and cobalt ions [4 ]. As a negative control, some sections were reacted with normal rabbit IgG instead of the specific antibodies.