Alexa flour 546 donkey anti rabbit igg

Immunostaining was conducted as previously described^{41 (link)}. After washing thrice with PBS/PVA, blastocysts were fixed with 3.7% formaldehyde at RT for 30 min. Next, blastocysts were permeabilized in 0.5% Triton X-100 for 30 min at RT, and incubated in PBS/PVA containing 3.0% BSA at RT for 1 h. Subsequently, these blastocysts were incubated overnight at 4 °C with rabbit anti-GRP78 (1:100; ab21685, Abcam, Cambridge, United Kingdom), rabbit anti-HSF1 antibody (1:100; 12972S, Cell Signaling, Danvers, MA, United States), and rabbit anti-caspase3 antibodies (1:20; 9664S, Cell Signaling). After washing thrice with PBS/PVA, the blastocysts were incubated with Alexa Fluor 488™ goat anti-rabbit IgG (1:200; A32731, Invitrogen) or Alexa Flour 546™ donkey anti-rabbit IgG (1:200; A10040, Invitrogen, Carlsbad, CA, United States) at 37 °C for 1 h. After three washes, the blastocysts were incubated for 10 min with 5 μg/mL Hoechst 33342. Finally, the blastocysts were mounted on slides and examined under a confocal microscope (LSM 710 META; Zeiss, Oberkochen, Germany). Images were processed using Zen software (v.8.0; Zeiss, Jena, Germany) and then analyzed using ImageJ v.l.44 g software (National Institutes of Health, Bethesda, MD, USA).

Immunofluorescent staining for RANKL and perilipin-1 was performed on paraffin sections of the left tibiae from middle-aged (13 months old) male mice. See Supplementary Methods for details. The following antibodies were used: anti-mouse perilipin-1 (#9349, Cell Signaling Technology, Danvers, MA, USA) (1:100), anti-mouse RANKL (#sc-7628, Santa Cruz Biotechnology, Dallas, TX, USA) (1:50), Alexa flour 546 donkey anti-rabbit IgG (#A10040, Invitrogen, Carlsbad, CA, USA) (1:100) and Alexa flour 488 donkey anti-goat IgG (#A11055, Invitrogen) (1:100).