Horse radish peroxidase secondary antibodies

Embryos were formaldehyde-fixed, and a standard immunohistochemical protocol was used for fluorescent- or DAB-visualized immunostaining as described previously [17 (link)]. The primary antibodies used were rabbit anti-Vasa (1:2000; gift of Paul Lasko), rat anti-Vasa (1:1000; gift of Paul Lasko), rabbit anti-pH3 (1:1000; Upstate Biotechnology), rabbit anti-CycB (1:500; gift of Jordan Raff), mouse anti-H5 to detect pSer2 (1:250; Research Diagnostics, Inc.), H3meK4 (1:500; gift of C. David Allis), rat anti-Twist (1:500; gift of Eric Wieschaus), rabbit anti-dpERK (1:100; Cell Signaling Technology), sheep anti-GFP (1:1,000; Bio-Rad), sheep anti-digoxigenin (DIG) (1:125; Roche), and mouse anti-biotin (1:125; Jackson Immunoresearch). Fluorescent immunostaining employed Alexa-Fluor secondary antibodies used at 1:500 (ThermoFisher), and DNA was labeled using either DAPI (10 ng/mL, ThermoFisher Scientific) or Hoescht (3μg/ml, Invitrogen). For DAB staining, horse radish peroxidase (HRP) secondary antibodies (Jackson Immunoresearch) were used 1:1000. Stained embryos were mounted using Aqua Poly/mount (Polysciences) on slides. At least three independent biological replicates were used for each experiment.

Immunoblotting was performed as described previously 38. The primary antibodies against acetylated histone H3 and β‐actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and antibodies against cleaved‐caspase 9, cleaved‐caspase 7, PARP, and BRCA1 were purchased from Cell Signaling Technology (Beverly, MA, USA). All horseradish peroxidase (HRP) secondary antibodies were purchased from Jackson Immuno Research Laboratories (West Grove, PA, USA).