Anti phh3

To determine the number of cells undergoing mitotic division, midguts were dissected and stained with anti-pHH3 (phospho-histone H3) antibody (Abcam, category no. ab14955). The number of pHH3-positive nuclei was counted from the entire midgut.

Antigen retrieval was performed using 0.01 M citrate buffer (pH 6.0) at 95°C for 20 min. The sections were processed for 0.25% Triton X‐100 treatment for 15 min, blocked with 5% goat serum in PBS for 1 h at room temperature (RT) and then incubated with corresponding primary antibodies (prepared in goat serum): anit‐RBM24 (Abcam, Cat no: ab94567, 1:300), anti‐Ki‐67 (Abcam, Cat no: ab15580, 1:300), anti‐pHH3 (Abcam, Cat no: ab5176, 1:300), anti‐PTEN (Santa Cruz, Cat no: SC‐7974, 1:300) overnight at 4°C. After washing five times with PBS, the sections were incubated with the appropriate secondary antibodies (prepared with goat serum) AlexaFluor^®488‐conjugated goat anti‐rabbit IgG (H+L) (Life Technologies, Cat no: A‐11008, 1:500), AlexaFluor^®488‐conjugated goat anti‐mouse IgG (H+L) (Life Technologies, Cat no: A‐11001, 1:500), or AlexaFluor^®555‐conjugated goat anti‐rabbit IgG (H+L) (Life Technologies, Cat no: A‐21428, 1:500) at RT in the dark for 1 h following washes with PBS. Next, slides were treated with the fluorescent blue dye Hoechst 33258 (Sigma, 1:1000) at RT for 10 min. After washing with PBS again, the slides were sealed with 50% glycerin. Representative pictures were photographed using a fluorescence microscope (Olympus, Japan).