Cy5 secondary antibody

Atrial appendages from non-T2D and T2D patients were fixed in 4% paraformaldehyde overnight (at 4°C) and paraffin embedded. Consecutive sections were cut (8 μm thick) and blocked with normal donkey serum (1 h) and immuno-labeled with monoclonal anti-caveolin-1 (Abcam, ab17052, 1:100, overnight, 4°C) antibody. Immuno-fluorescent labeling was performed with corresponding Cy5 secondary antibody (Jackson ImmunoResearch, West Grove, PA, United States). DAPI was used for nuclear staining. For non-specific binding, the primary antibody was omitted. Structured illumination microscopy (SIM-Apotome, AxioImagerM2, CarlZeiss, Jena, Germany) was used for immunofluorescent detection.

The following antibodies were used for immunoblot (IB) and immunofluorescence (IF) experiments: Human anti‐ACA (1:30, Antibodies Incorporated #15‐234), Mouse anti α‐Tubulin Alexa Fluor 488 conjugated (IF:1:100, Life Technologies #322588), Rabbit anti‐ MAD2 (IF:1:1000, IB: 1:500; Biolegend #924601 or previously Covance #PRB‐452C)), sheep anti‐BUB1 (1:100, gift from Dr. S. Taylor), rabbit anti‐MPS1 (1:100, gift from Dr. H. Yu), rabbit anti‐Securin (IB: 1:500, Invitrogen #700791) rabbit anti‐ZW10 (IF:1:100; IB:1:500, Abcam #ab21582), rabbit anti‐HEC1 (1:100; gift from Dr. R. Benezra), rabbit anti α‐Tubulin WB:1:500 Cell Signaling Technology #11H10). The following secondary antibodies were used at 1:200 for IF experiments: goat‐anti‐human‐Alexa‐633 (Life Technologies #A21091), donkey‐anti‐rabbit‐Alexa‐568 (Life Technologies #A10042) and Cy5 secondary antibody pre‐absorbed against goat serum proteins (Jackson Immunoresearch). Monastrol (Sigma #M8515), and nocodazole (Sigma #M1404) were dissolved in dimethyl sulfoxide (DMSO) (Sigma) and added to the CZB culture media at final concentrations of 100 μM and 5 μM, respectively. N‐Acetyl‐L‐Cysteine (NAC) (Abcam #Ab143032), ROS inhibitor, was dissolved in embryo water and added to CZB at final concentration of 5 mM. In vitro maturation of drug‐treated oocytes was performed in organ culture dishes.

Internalization assays were performed as described6 (link), with minor modifications. Briefly, hippocampal neurons at 15–18 days DIV were incubated with antibodies against the N-terminus of GluA2 (2 μg/ml, mouse monoclonal, clone 6C4, Millipore) for 30–60 min at 18–20 °C. After brief washing, neurons were either incubated with control medium or medium containing NMDA (50 μM) for 15 min at 37 °C. Subsequently, they were fixed for 5 min at room temperature in paraformaldehyde (4%)/sucrose (4%) without permeabilization. Surface-remaining antibody-labeled receptors were visualized by means of a 1-h incubation with saturated Cy5-secondary antibody (15 μg/ml, Jackson ImmunoResearch). Neurons were permeabilized for 2 min with methanol (−20 °C), and internalized antibody-labeled GluA2 was detected by a 1-h incubation with Alexa 568-conjugated secondary antibody (1 μg/ml, Invitrogen). Simultaneously, infected GFP-fluorescent neurons were stained with antibodies against GFP (5 μg/ml, Invitrogen, rabbit polyclonal). Thus, the GFP fluorescence of infected neurons was enhanced by a 30 min incubation with Alexa 488-conjugated secondary antibodies (4 μg/ml, Invitrogen). After staining, the coverslips were mounted in Mowiol (Sigma-Aldrich, Barcelona, Spain).