Goat anti rabbit alexafluor633

All reagents were purchased from Sigma Aldrich unless otherwise noted. Cell culture media was obtained from ThermoFisher Scientific and Lipidure®-CM from AMS Biotechnology (Europe) Ltd. The enzymes used for cell isolation were collagenase Type F, collagenase Type 3 (Worthington, NJ, USA), papain (Worthington) and hyaluronidase. Cell culture dishes with glass coverslip bases (Ibidi µ-Dish 35 mm, high) were purchased from Thistle Scientific (UK). Tali™ Apoptosis Kit (Annexin V Alexa Fluor™ 488) and Dil-conjugated oxLDL from Human Plasma (Dil-OxLDL) were both from Invitrogen™ (ThermoFisher Scientific). The antibodies used for immunocytochemistry were mouse anti-SMA-Cy3 (C6198, Sigma-Aldrich), rabbit anti-Galectin 3 (PA579595 ThermoFisher Scientific) and goat anti-rabbit-AlexaFluor633 (A21071, ThermoFisher Scientific), along with Hoechst33342 Solution (ThermoFisher Scientific).
The buffers used during cell isolation were: Mops buffer (145mM sodium chloride, 2mM MOPS, 4.7mM potassium chloride, 1.2 mM monosodium phosphate, 5mM glucose, 0.02 mM EDTA, 2mM sodium pyruvate, 1.2mM magnesium chloride, 2mM calcium chloride, pH 7.4) and isolation buffer (80mM sodium glutamate, 55mM sodium chloride, 6mM potassium chloride, 10mM glucose, 10mM Hepes,1mM magnesium chloride, 0.1mM calcium chloride, 0.2mM EDTA, pH 7.4), with or without 2 mg/ml fatty acid free bovine serum albumin (BSA).

The following antibodies were purchased from Cell Signaling Technology: rabbit anti-IRS-1 (2382), rabbit anti-pAKT S473 (9271), rabbit anti-p85 (4292), rabbit anti-Grb2 (3972), rabbit anti-FoxO1 (2880), rabbit anti-ERK1/2 (9102), rabbit anti-pERK1/2 (4370), anti-IGF-1Rβ (9750), anti-IRβ (3025), anti-pIGF-1Rβ Y1135/1136/pIRβ Y1150/1151 (3024), anti-pIGF-1Rβ Y1131/pIRβ 1146 (3021). Other antibodies were from the following commercial sources: rabbit anti-pIRS-1 Y612 (Invitrogen, 44-816 G); rabbit anti-AKT (HUABIO, ET1609-47), rabbit anti-GFP (HUABIO, ET1607-31), mouse anti-Actin (HUABIO, M1210-2), mouse anti-MHC (DSHB, MF20), mouse anti-FLAG (YEASEN, 30503ES60), and mouse anti-mCherry (ABclonal, AE002). HRP-conjugated secondary antibodies: goat anti-mouse second antibody (Jackson, 115-035-003) and goat anti-rabbit second antibody (Jackson, 111-035-003). Fluorescent secondary antibodies were from Thermo Fisher Scientific: goat anti-mouse-Alexa Fluor 546 (A-11003), goat anti-rabbit-Alexa Fluor 488 (A-11008), goat anti-rabbit-Alexa Fluor 546 (A-11010), goat anti-rabbit-Alexa Fluor 633 (A-21071).

Control 2/16 pairs of blastomeres and experimental embryo fragments (2/16 pairs and 4/32 quartets) were fixed in 4% PFA for 40 min at room temperature, and then permeabilized with 0.3% Triton X-100 for 15 min. The nonspecific antibody binding was blocked by incubation in 3% BSA (for CDX2 staining) or in 10% FBS (for p-Ezrin staining), overnight, at 4°C. Embryos and embryo fragments were then incubated with primary antibodies (mouse monoclonal anti-CDX2 antibody (1:50, BioGenex, USA) or rabbit monoclonal antibody anti-p-ERM (phospho-Ezrin(Thr567)/Radixin(Thr564)/Moesin(Thr558), 1:400, Cell Signaling Technology, 3141) at 4°C, overnight, and then treated with secondary antibodies (TRITC-conjugated goat anti-mouse antibody (1:200, Jackson ImmunoResearch) or goat anti-rabbit AlexaFluor 633 (1:200, Molecular Probes)) for 1 hour at room temperature. Additionally, chromatin was stained by incubation with DRAQ5 (10 μM; Biostatus, Leicestershire, UK) or chromomycin (0.01 mg/ml) for 10 min at 37°C, and F-actin by incubation with FITC-conjugated phalloidin (1:1000) for 20 min at room temperature. The embryos and embryos fragments were analyzed using a Zeiss 510 confocal laser microscope.