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?
Specimen
Type:
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?
Staining Protocol
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.