Alexafluor 546 conjugated donkey anti mouse

Briefly, cultured rat hippocampal neurons were washed twice with PBS containing (mM) 1 MgCl₂ and 2 CaCl₂. Neurons were fixed with 4% paraformaldehyde for 20 min at room temperature (RT) and washed 3 times after fixation. Then neurons were permeabilized with PBS containing 0.1% triton x-100 and 2 mg/ml BSA for 30 min. Neurons were blocked with PBS containing 4% milk and 2 mg/ml BSA for 2 h at RT. Cav3.2 (mouse anti-Cav3.2, 1 μg/ml, Novus Biologicals,CA) primary antibodies were incubated overnight at 4 °C. After washing three times, the secondary antibodies Alexafluor 546 conjugated Donkey anti-mouse (Thermo Fisher Scientific, CA) were incubated 2 h. All images were digitally captured with an 8 bit camera, thus giving grey level (intensity) values of 0–255. Immunostaining was visualized using a 40 × 0.4 NA objective lens on a Zeiss LSM 510 META confocal systems, running Velocity 6.

Cells were washed with PBS (Nacalai Tesque) twice, fixed with 4% PFA (Nacalai Tesque)/PBS for 20 min at 4 °C and blocked in PBS with 5% donkey serum (Millipore) and 0.4% Triton X-100 (Nacalai Tesque) (blocking solution) for 30 min at room temperature. The following primary antibodies were diluted in blocking solution and incubated overnight at 4 °C: goat anti-PDX1 (R&D Systems, AF2419, 1:200), mouse anti-NKX6.1 (DSHB, F55A12, 1:200), rabbit anti-NKX6.1 (Cell Signaling, 54551, 1:500) and rat anti-CPEP (DSHB, GN-ID4, 1:200). After washing with PBS, the cells were incubated with the following fluorescent secondary antibodies for 1 h at room temperature: Alexa Fluor 488-conjugated donkey anti-goat (Thermo Fisher Scientific, A-11055, 1:500), Alexa Fluor 546-conjugated donkey anti-mouse (Thermo Fisher Scientific, A-10036, 1:500), Alexa Fluor 555-conjugated donkey anti-rabbit (Thermo Fisher Scientific, A-31572, 1:500) and Alexa Fluor 488-conjugated donkey anti-rat (Thermo Fisher Scientific, A-21208, 1:500). Immunofluorescence images were acquired using a CellInsight NXT (Thermo Fisher Scientific) or BZ-9000 (Keyence). For quantification, 9 images per well were obtained using CellInsight NXT.