Immunofluorescence Technical Support
NOTE: For the best possible results, Cell Signaling Technology (CST) strongly recommends using our optimized application-specific protocols for each product. These protocols are the result of extensive in-house validation performed at CST and ensure accurate and reproducible results.
To ensure a timely solution, please have the information below ready when you contact us. Technical Support can be reached by toll free phone at 877-678-8324 or 877-678-TECH, fax at 978-867-2400 or e-mail at firstname.lastname@example.org.
- Company/university name.
- Type of microscope/imaging system.
- Excitation source (specific lasers, mercury bulb, etc).
- Catalog number.
- Lot number (printed on tube label).
- Dilution you are using.
- Secondary antibody (if not using conjugated antibody).
- Secondary vendor/catalog number.
- Fluorochrome (eg. FITC).
- Secondary dilution.
- Have you successfully used this secondary antibody for immunofluorescence?
- ICC/cell line
- Paraffin section
- Fixed frozen section
- Fresh frozen section
- Specimen source (cell line or tissue, species).
- How were the cells/tissues treated prior to fixation (ligands, inhibitors, etc.)?
- Fixation method (immersion, perfusion).
- Description of fixation and specimen prep protocol, including type of fixative, incubation times and concentrations.
- If using cell lines, how much time elapsed after the cells were treated and before fixation?
- How much time elapsed after sectioning/fixation and staining?
- How were unstained sections stored?
- Antigen Retrieval.
- Blocking Method.
- Dilution buffer (including detergents).
- Time/temperature of primary antibody incubation.
- Time/temperature of secondary antibody incubation.
- Please list other primary antibodies used on same slide.
- Are you certain that the target is present in these cells/tissue?
- If you using a phospho antibody, has this experimental protocol been shown to affect phosphorylation of this target (confirmed by published references or other application)?
- Positive control (treated cells, alternate tissues, etc.).
- Negative control:
- No primary
- Isotype control
- Negative cells/tissue
- Ig concentration of isotype control antibody, if applicable.