Immunostaining of Histone H4 and Chromatin

Lam-KD, LBR-KD or control S2 cells were seeded on coverslips for 30 min. After rinsing with PBS, cells were fixed in 100% methanol for 5 min at room temperature (for further examination of chromatin distribution based on the immunostaining of histone H4) or in 4% formaldehyde in PBS for 25 min at room temperature (for further estimation of chromatin volume based on DAPI staining), rinsed with PBS three times, blocked with PBTX (PBS with 0.1% Tween-20 and 0.3% Triton X-100) containing 3% normal goat serum (Invitrogen) for 1 h at room temperature. The remaining immunostaining procedure was performed as previously described^{50 (link)}. As primary antibodies we used murine monoclonal anti-histone H4 (1:200; ab31830, Abcam), guinea pig polyclonal anti-LBR^{26 (link)} (1:1000), rabbit polyclonal anti-lamin Dm0^{51 (link)} (1:500). As the secondary antibodies we used Alexa Fluor 546-conjugated goat anti-rabbit IgG (Invitrogen) or Alexa Fluor 488-conjugated goat anti-mouse IgG (Invitrogen), or Alexa Fluor 633-conjugated goat anti-guinea pig IgG (Invitrogen).