Cy5 conjugated donkey anti mouse antibody

The following primary antibodies were used for immunofluorescence labeling or immunoblotting: mouse anti-ACBD3 (Sigma) at a 1:1,000 dilution for immunofluorescence labeling and a 1:3,000 dilution for immunoblotting; mouse anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH) (Abcam) at a 1:1,000 dilution and mouse anti-Flag (M2; Sigma) at a 1:2,000 dilution for immunoblotting; rabbit antigiantin (Covance) at a 1:1,000 dilution, rabbit anti-TGN46 (LifeSpan Biosciences) at a 1:500 dilution, and goat anti-Salmonella antibody (CSA-1; Kirkegaard & Perry Laboratories) at a 1:200 dilution for immunofluorescence labeling; and mouse anti-HA (HA.11; Covance) at a 1:5,000 dilution for immunoblotting and a 1:1,000 dilution for immunofluorescence labeling.
Rhodamine Red X-conjugated donkey anti-mouse or anti-rabbit antibody, donkey anti-goat or donkey anti-rabbit–cyanine 2 (Cy2) antibody, and Cy5-conjugated donkey anti-mouse antibody were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA), for immunofluorescence labeling. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit and goat anti-mouse antibodies were purchased from Dako for immunoblotting.

To verify that DREADD expression was limited to ACA CaMKII-expressing neurons, sections containing frontal cortex and striatum from all animals receiving DREADD were immunostained for mCherry and CaMKII and sections throughout the brain stained for mCherry. Sections were incubated overnight with a rabbit antibody specific for mCherry (1:4,000; Abcam prod. # ab167453, Cambridge, MA; RRID:AB_2571870) and mouse CaMKII antibody (1:1,000; Thermo Fisher Scientific), followed by incubation with Dylight 488 goat anti-rabbit antibody (1:100; 30 min; Thermo Fisher Scientific prod. # 35553; RRID:AB_1965947) and Cy5-conjugated donkey anti-mouse antibody (1:100; 30 min; Jackson Immunoresearch Laboratories). mCherry immunostaining in cells was exclusively co-localized with endogenous red fluorescence indicating mCherry, as previously shown (Beloate et al., 2016 (link)) and further validating the antibody. CaMKII antibody has previously been validated (Fanous et al., 2013 (link); Kuiper et al., 2017 (link)).

Cells grown on cover slips were washed in PBS and fixed in methanol:acetone [1∶1] at −10°C for 15 minutes. After a wash in PBS, samples were blocked at RT for 30 minutes in PBS containing 10% horse serum. After a wash in PBS, samples were incubated at 4°C overnight with the primary antibody in PBS containing 2% horse serum. After three 5 minutes washes in PBS, samples were incubated at RT for 1 hour with the secondary antibody in PBS containing 2% horse serum. After three 10 minutes washes in PBS, samples were mounted in Vectashield hard containing DAPI. Images were visualized on a Zeiss 510 Meta Confocal Laser Scanning Microscope. Primary antibodies used were a rabbit anti-TRF2 (H-300, Santa Cruz) and mouse anti-γ-H2AX antibodies (clone JBW301, Upstate). Secondary antibodies were a FITC-conjugated donkey anti-rabbit antibody and a Cy5-conjugated donkey anti-mouse antibody, both from Jackson ImmunoResearch.