Alexa 594 goat anti mouse

To investigate the potential role of Ca_v1.3 in phospholamban (PLB) phosphorylation on serine 16 or threonine 17, isolated SANC needed to be in a physiological solution. Therefore, immunostaining was completed after Ca²⁺ reintroduction (see single cell isolation protocol above). SANC were plated on a multi-well glass slide (see above) and incubated for 5 min in Tyrode solution. Cells were separated in three groups and incubated with: condition 1: Tyrode for 6 min; condition 2: 100 nM isoproterenol (ISO) for 6 min; or condition 3: 100 nM ISO for 3 min and then ISO + 3 µM nifedipine (Nife) for 3 min. Immunostaining was then performed as described above with anti-PLB total (mouse, 1:200, Badrilla, Cat. No. A010-14) and anti-phosphorylated PLB at serine 16 (rabbit, 1:200, Badrilla, Cat. No. A010-12AP) or anti-phosphorylated PLB at threonine 17 (rabbit, 1:200, Badrilla, Cat. No. A010-13) primary antibodies and Alexa 594 goat anti-mouse (1:200, Jackson laboratories, Cat. 115-585-003) and Alexa 488 goat anti-rabbit (1:200, Thermo Fischer Scientific, Cat. A11034) secondary antibodies.

Neurons were fixed for 20 min at room temperature (RT) in 4% paraformaldehyde prewarmed to 37°C, rinsed and permeabilized for 6 min with 0.12% Triton-X plus 0.12% gelatin in phosphate-buffered saline (PBS), blocked 1 h at RT in PBS/gelatin, and incubated for 1 h at RT with anti-microtubule associated protein 2 (MAP2) mouse monoclonal antibody (diluted 1:200 in PBS/gelatin; Sigma-Aldrich) and with anti-Tau rabbit polyclonal antibody (diluted 1:500 in PBS/gelatin; Abcam, United Kingdom). After rinsing with PBS, cells were incubated for 1 h at RT with the secondary antibodies Alexa 594 goat anti-mouse for the detection of MAP2, and Alexa 488 goat anti-rabbit, for the detection of Tau (diluted 1:1,000 in PBS/gelatin; Jackson ImmunoResearch, United States). After washing with PBS, glass coverslips were mounted on slides with Vectashield antifade mounting medium containing DAPI (Vector Laboratories, Burlingame, CA, United States).

The following primary antibodies were used: mouse anti-Tubulin beta III isoform C-terminus, clone TUJ1 (1:500; Millipore; RRID:AB_2210524), goat anti-Oncomodulin (Ocm) antibody (1:1000; Santa Cruz; RRID:AB_2267583), rabbit anti-Foxo3 (1:1000; Thermo Scientific; RRID:AB_2544621), rabbit anti-Myosin7a (Myo7a) (1:200; Proteus; RRID:AB_10013626) mouse anti-Ctbp2 (aka C-Terminal Binding Protein 2; 1:200; BD Transduction Laboratories; RRID:AB_399431), and mouse anti-Gria2 (aka GluR2/GluA2; 1:2000; Millipore; RRID:AB_2113875). The following secondary antibodies, all purchased from Jackson ImmunoResearch, were used: Donkey Anti-Mouse 488 (1:500; RRID:AB_2340849), Donkey Anti-Rabbit 594 (1:500; RRID:AB_2340622), Donkey Anti-Rabbit 647 (1:200; RRID:AB_2340625), Donkey Anti-Goat 647 (1:200; RRID:AB_2340438), Alexa 594 Goat Anti-Mouse (IgG1, 1:500; RRID:AB_2338885), Alexa 488 Goat Anti-Mouse (IgG2a, 1:500; RRID:AB_2338855).