Alexa fluor fluorescent secondary antibody

For doing western blots in Drosophila, the following primary antibodies were used: anti-FUS (Bethyl Laboratories; A300-302A, 1:2000), anti-MBNL1 (Millipore; MabE70, 1:2000), and anti-mbnl1 (Invitrogen; MA1-16967, 1:1000). DyLight fluorescent secondary antibodies (Thermo Fisher Scientific) were used at a concentration of 1:10,000
For the Drosophila NMJ analysis, the following primary antibodies were used: Alexa Fluor 488-conjugated goat anti-HRP (Jackson Immuno Research; 123-545-021, 1:200) and mouse anti-DLG 4F3 (DSHB, 1:100). The following secondary antibodies were used: Alexa Fluor 647-conjugated anti-Phalloidin (Invitrogen; A22287, 1:250) and goat antimouse Alexa Fluor 546 (Invitrogen, A11030, 1:500).
The following primary antibodies were used: anti-G3BP1 (Protein Tech; 13057-2-AP, 1:2000) and anti-HA7 (Sigma, H3663, 1:500). Alexa-Fluor fluorescent secondary antibodies (Invitrogen) were used at a concentration of 1:500 for doing Western blots in HEK293 and N2a cell lines.
The following primary antibodies were used for doing immunoblotting in primary neuronal cells: anti-MAP2 (EMD Millipore; AB5622, 1:500), anti-FUS (Proteintech; 11570-1-AP, 1:200), anti-TIA1 (Santa Cruz; SC-1751, 1:250), anti-G3BP1 (Proteintech; D5444, 1:100), anti-MBNL1 (Millipore; MABE70, 1:100), and anti-NEFL (Novus Biologicals; NB300-222, 1:300).

S. purpuratus embryos were collected at the desired time point and fixed for 5–10 min in 4% paraformaldehyde in PEM buffer [92 (link)]. Embryos were washed with phosphate-buffered saline (PBS), blocked for 1 h in SuperBlock (Thermo), probed with primary antibody, and washed 3 times with PBS. Alexa Fluor fluorescent secondary antibodies (Invitrogen) were used to visualize antibody labeling on a Zeiss 700 LSM (Carl Zeiss) confocal microscope. All preparations were done at 4 °C. Imaging and analysis were conducted using ZEN (2009) or ImageJ (1.44) software. Adobe Photoshop (9.0.2) was used to prepare the figures and adjust image contrast and brightness. Antibodies deployed here: anti-SynB [43 (link)], Sp1 [46 (link)], and anti α-tubulin (Santa Cruz Biotechnologies, sc-23948).

The following reagents were used for immunofluorescence: rat anti-platelet endothelial cell adhesion molecule 1 (PECAM1; BD Pharmingen, 553370, 1:250-1000), mouse anti-α-smooth muscle actin-Cy3 (Sigma-Aldrich, C6198, 1:100-1000), rat anti-E-cadherin (Invitrogen, 53-3249-82, 1:500), rabbit anti-phosphohistone H3 (EMD Millipore, 06-570, 1:250) mouse anti-HOPX (E-1; Santa Cruz Biotechnology, sc-398703, 1:100), rabbit anti-SPC (Abcam, ab90716, 1:500). Prolong Gold mounting media (±DAPI) and Alexa Fluor fluorescent secondary antibodies (Alexa 488 donkey anti-rat A21208, Alexa 594 donkey anti-rabbit A21207, both diluted to 1:1000) were purchased from Invitrogen. The nuclear stain DRAQ5 (62251, 1:5000) was purchased from Thermo Fisher Scientific. Antibody titrations were performed on WT tissues to determine the optimal dilution for each tissue. Cells and explants were cultured in the following reagents: interferon-γ (R&D Systems, 485-MI-100), Dulbecco's modified Eagle's medium (DMEM; Corning Life Sciences, 10-013-CV), fetal bovine serum (FBS; Thermo Fisher Scientific), and penicillin-streptomycin (Thermo Fisher Scientific, 15140122).

OX1-19 iPSC-derived neurons were cultured to day 80 on coverslips and fixed in 4% paraformaldehyde (PFA). Coverslips were blocked in 10% donkey serum (Sigma #D9663). Antibodies against MAP2 (Abcam #ab92434) and βIII-Tubulin (Abcam #ab18207) were used. Coverslips were then incubated in Alexa-fluor fluorescent secondary antibodies (ThermoFisher), mounted on slides using prolong gold containing 4′,6′-diamidino-2-phenylindole (DAPI) (358nm absorbance) (Cell Signalling Technology #8961) and imaged on an EVOS® FL Cell Imaging System (ThermoFisher).

HEK293T cells were seeded onto 18-mm coverslips coated with poly-L-lysine and transiently transfected with OR constructs with or without accessory proteins RTP1s and Ric8b (Lipofectamine 2000, Invitrogen). Flag-tagged OR trafficking was assayed using surface immunofluorescence as previously described (Shepard et al., 2013 (link)). Briefly, live, non-permeabilized cells at 4°C were exposed to a rabbit polyclonal anti-Flag antibody (Sigma) in 0.1% BSA/PBS. This antibody will only detect the extracellular Flag epitope of those receptors that are functionally expressed on the plasma membrane. Subsequently, cells were washed, fixed with 4% paraformaldehyde, permeabilized (0.3% Triton X-100), and then exposed to a mouse monoclonal (M2) anti-Flag antibody (Sigma). As the external Flag epitope (surface Flag) is ‘blocked’ after binding to the polyclonal Flag antibody, the monoclonal Flag antibody detects only the internal population of ORs. Alexa Fluor fluorescent secondary antibodies (Thermo Fisher) were used to detect the localization of the polyclonal and monoclonal Flag-tags. Slides were imaged using the ZEISS Axiovert 200 m microscope at 40× magnification.