Immunofluorescence Technical Support

To ensure a timely solution, please have the information below ready when you contact us.

General Questions

1. Company/university name.
2. Type of microscope/imaging system.
3. Excitation source (specific lasers, mercury bulb, etc).

Antibody Information

1. Catalog number.
2. Lot number (printed on tube label).
3. Dilution you are using.
4. Secondary antibody (if not using conjugated antibody).
5. Secondary vendor/catalog number.
6. Fluorochrome (eg. FITC).
7. Secondary dilution.
8. Have you successfully used this secondary antibody for immunofluorescence?

Specimen

1. Type:
   1. ICC/cell line
   2. Paraffin section
   3. Fixed frozen section
   4. Fresh frozen section
2. Specimen source (cell line or tissue, species).
3. How were the cells/tissues treated prior to fixation (ligands, inhibitors, etc.)?
4. Fixation method (immersion, perfusion).
5. Description of fixation and specimen prep protocol, including type of fixative, incubation times and concentrations.
6. If using cell lines, how much time elapsed after the cells were treated and before fixation?
7. How much time elapsed after sectioning/fixation and staining?
8. How were unstained sections stored?

Staining Protocol

1. Antigen Retrieval.
2. Blocking Method.
3. Dilution buffer (including detergents).
4. Time/temperature of primary antibody incubation.
5. Time/temperature of secondary antibody incubation.
6. Please list other primary antibodies used on same slide.
7. Are you certain that the target is present in these cells/tissue?
8. 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)?
9. Positive control (treated cells, alternate tissues, etc.).
10. Negative control:
    1. No primary
    2. Isotype control
    3. Negative cells/tissue
11. Ig concentration of isotype control antibody, if applicable.