Troubleshooting

If something doesn’t work check through the following list to resolve the problem. If there are still difficulties and you have purchased an AbD Serotec antibody our Technical Services Team will be happy to offer further advice.

Problem Course of action
No staining
  1. Ensure that all antibodies have been stored correctly according to the manufacturer’s instructions.
  2. Ensure that commercial antibodies have not exceeded their date of expiration.
  3. Ensure that appropriate primary or secondary antibodies have been added.
  4. Ensure that antibody is conjugated to a fluorochrome. If not, ensure that appropriate fluorochrome-conjugated secondary is being used.
  5. Ensure that secondary antibody is active – has it been used successfully with other primary antibodies?
  6. Ensure that correct secondary antibody is being used, which will recognize your primary antibody.
  7. If the fluorochrome used is Phycoerythrin or Allophycocyanin based, ensure that the product has not been frozen.
  8. Is the target antigen present on test tissue? Check literature for antigen expression and incorporate a positive control of known antigen expression alongside test material.
  9. Does antibody recognize antigen in test species? Check that antibody cross-reacts with species being used. Not all antibodies will cross-react across species.
  10. Ensure that correct laser is being used to excite fluorochrome, and that correct channel is being used to analyze emissions.
PE antibody does not stain but same FITC antibody gives good results
  1. PE conjugate may have been frozen. If so, purchase another vial of antibody.
  2. Paraformaldehyde (PFA) may be a problem. Breakdown of PFA may release methanol, which will affect staining. Make up fresh paraformaldehyde. Cells can be analyzed immediately without fixing.
Non-specific staining
  1. Non-specific staining may be due to autofluorescence. Solution: check levels of autofluorescence by including a tube of cells only (i.e. without any antibody) into your panel.
  2. Certain cells express low affinity Fc receptors CD16/CD32, which bind whole antibodies via Fc region. For mouse cells, dilute antibody in SeroBlock FcR (Product Codes BUF041 A/B).
  3. Non-specific staining may be due to the secondary antibody. Select a secondary antibody that will not cross-react with target tissue.
  4. Ensure that sufficient washing steps have been included.
  5. Titrate test antibody carefully. Non-specific staining may be reduced at lower antibody concentrations.
Weak staining
  1. Weak staining may be due to overdilution of antibodies. Ensure that antibodies are used at the correct concentration by titrating antibodies before use.
  2. Weak staining in indirect staining systems may be due to prozoning effect, where highly concentrated antibodies may give weak results. Titrate antibodies carefully.
  3. Weak staining may be due to an excess cell number. Adjust cell population to recommended density.
  4. Weak staining may be due to the antigen expression. Check literature for expected levels of expression.
  5. If antigen expression is weak, select an antibody that is conjugated to a brighter fluorochrome.
  6. Weak staining may be seen if using a cross-reacting antibody rather than one specific for the target species.
  7. Incubation time and temperature with either primary or secondary antibody should be optimized.
Unusual scatter profiles
  1. Ensure that cells are used as fresh as possible. Profile may be showing dead cells and debris.
  2. Activation methods may affect scatter characteristics of cells.
  3. If you are using lysing solution, ensure that this is fresh and has been made up correctly.
Unexpected staining
  1. Some reagents may affect certain antigens and, therefore, may need reviewing e.g. EDTA will affect some platelet markers.
  2. Lysing solutions may affect certain antigens. Select a method that does not interfere with antigen detection.
  3. Some antigens are expressed intracellularly and, therefore, cell permeabilization methods may be required. Check manufacturer’s datasheet for correct permeabilization reagent.

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