Apex antibody labeling kit

Cells grown on coverslides were fixed and permeabilized with 100% methanol for 5 min and non-specific binding sites were blocked with 1% albumin for 1 h at 37°C. Then, cells were incubated overnight at 4°C with the α-H4R3me2 (1:50) or α-PRMT1 (1:20) antibodies. After several washes, samples were incubated with an anti-rabbit IgG secondary antibody conjugated to FITC (Zymed) (1:100). For co-immunolocalization, α-H4R3me2 and α-PRMT1 antibodies were covalently labeled with Pacific blue 410 and Alexa fluor 647, respectively, using the APEX Antibody Labeling Kit (Life Technologies) according to the manufacturer’s recommendations. Then, cells were incubated overnight at 4°C with these antibodies.
Nuclei were stained with 4′,6-Diamidino-2-Phenylindole (DAPI) and samples were observed through a confocal microscope (Carl Zeiss LSM 700) using the ZEN 2009 software. Observations were performed in approximately 20 optical sections from the top to the bottom of each sample.

Both neuropeptide antibodies (anti-FMRFamide and anti-ATR) and the MsHisClB receptor antibody were produced in rabbit hosts. Therefore, to double label using the neuropeptides and the rabbit anti-MsHisClB antibodies we directly fluorescently tagged each primary antibody using the APEX antibody labeling kit (Life Technologies, A10468 488, A10475 for 647; Woo et al., 2010 (link)). This method covalently bonds the IgG antibody to a fluorescent label, and therefore eliminates cross reactivity of secondary antibodies with primary antibodies raised in the same animal. To remove contaminants, the labeling tip was hydrated with 100 μL of wash buffer to which 10–20 μg of IgG antibody is added and eluted with a syringe: 10 μL of MsHisClB, and 1 μL of both FMRFamide and ATR antibody, respectively. This solution was then combined with reactive dye (either Alexa 488 or Alexa 647) containing 2 μL of DMSO and 18 μL of labeling buffer. This solution then incubated for 2 h at room temperature. The solution was washed with 50 μL of buffer and eluted through the tip. Finally, 40 μL of elution buffer is eluted through the tip and mixed with 10 μL of neutralization buffer to yield a final volume of ∼50 μL of solution. This solution was then diluted in 350 μL of PBSAT and tissue was incubated for 48 h at 4°C.