Alexa fluor 488 conjugated donkey anti rabbit igg

Human KC were harvested and stained with fixable viability dye eFluor 780 (eBioscience; now Thermo Fisher Scientific, Carlsbad, USA) according to the manufacturer instructions. After fixation in 2% paraformaldehyde, permeabilization was achieved using 0.5% saponin in PBS with 3% FBS and 0.1% sodium azide. The K1-specific antibody was used in combination with Alexa Fluor 488-conjugated donkey anti-rabbit IgG (Dianova). All incubation steps were performed for 30 minutes at RT. The cells were acquired on a BD LSRFortessa II (BD Bioscience, Heidelberg, Germany) and analyzed using FlowJo software (Flow Jo LLC, Ashland, Oregon)

Apoptosis was induced by treatment with either staurosporine (Sigma, #S4400), ABT-737 (Selleck Chemicals, #S1002), Mcl-1-inhibitor S63845 (APExBio # A8737), the combination of ABT-737 and the Mcl-1-inhibitor S63845, or the combination of Bcl-X_L inhibitor A‑1155463 (Biozol, #S7800) and the Mcl-1-inhibitor S63845. Cells were collected, washed and fixed with 4% paraformaldehyde (Morphisto, #11762.00250) for 30 min at room temperature, followed by staining with anti-active caspase-3 antibody (BD Pharmingen, #559565) in PBS (Life Technologies, #14190169) containing 0.5% Saponin (Roth, #4185.1) and 0.5% bovine serum albumin (BIOMOL, #BSA-50). Alexa Fluor 647-conjugated donkey anti-rabbit IgG (Dianova, #711605152) or Alexa Fluor 488 conjugated donkey anti-rabbit IgG (Dianova, #711-545-152) were used as secondary antibody and cells were analyzed by flow cytometry using a FACS Calibur Flow Cytometer (Becton-Dickinson, Heidelberg), in combination with the software FlowJo version 10.4.

Spreads of Sd. ludwigii meiotic nuclei were prepared from sporulating cells in meiotic time courses using the procedure previously described for S. cerevisiae [144 (link)] with some modifications. The detailed protocol is provided in the Additional file 10: Supplementary detailed methods. The primary antibodies used were as follows: rabbit anti-ScRfa1 (gift from E. Mancera), sheep anti-GFP (generated in our lab) at 1:500 dilution each, and rabbit anti-ScRad51 (gift from A. Shinohara) at dilution 1:1000. The latter antibody [145 (link)] was used for detection of discrete Rad51 foci, while the anti-GFP antibody was used for localization studies of GFP-tagged Rap1 in spreads of meiotic nuclei. The secondary antibodies used were as follows: Alexa Fluor 488-conjugated donkey anti-rabbit IgG (Dianova), Cy2-conjugated donkey anti-sheep IgG (Dianova) and Cy3-conjugated donkey anti-rabbit IgG (Dianova), at 1:500 dilution each.