To ensure a timely solution, please have the information below ready when you contact us.
Please Provide The Following Information
- Which CST antibody are you using (target, dilution, lot number and reference date)? What secondary antibody are you using (vendor, fluorochrome, dilution)?
- Have you successfully used this secondary before?
- What type of cells are you using (whole blood, PBMCs, cell line, marrow, dissociated tissue, ...)?
- Briefly describe the fixation/permeabilization protocol (specifically, did you use methanol to permeabilize or did you use a kit?).
- Do you need to use other marker/gating antibodies at the same time (CD45, cytokeratin, etc.)?
- How were these cells stimulated/inhibited? (please address the following questions in your answer)
- Are you certain that the target is present in these cells?
- If you using a phospho antibody, has this experimental protocol been shown to affect phosphorylation of this target?
- Do you have a positive/negative control (serum starved, specific inhibitor, ligand activated, etc.)?
- How much time elapsed after the cells were treated and before fixation?
Major Reasons For No Signal
- Using wrong laser/PMT with fluorochrome.
- Inadequate fixation and permeabilization (most antibodies directed against intracellular antigens will not work with commercial fix & perm kits that use a detergent for permeabilization).
- Stimulation did not induce phosphorylation of desired target.
- Target was dephosphorylated (waiting too long between stimulation and fixation, eg. incubating with CD's before fixation).