FAQ

Troubleshooting

Frequently asked questions

• How do I resuspend my lyophilised antibody?
• How stable are the diluted antibodies?
• Do I need to freeze my antibody aliquots?
• How long should I incubate the tissue/cells with the primary antibody?
• How should I fix my tissue/cells?
• Do I need to permeabilize the cells?
• Should I use frozen or paraffin sections?
• Immunohistochemistry (IHC) or immunofluorescence (IF/ICC)? Which is the most sensitive method?
• Alkaline Phospatase (AP) or Peroxidase (HRP)? Which is the most sensitive?
• How can I reduce the background signal in IHC or IF/ICC?
• I cannot see the staining in IHC or IF/ICC, do you have some tips?
• I have non-specific staining, do you have some tips?
• Why do I need to use a positive control and a negative control?

How do I resuspend my lyophilised antibody?

Reconstitute in an equivalent volume to the weight you purchased, so that the final concentration is 1 mg/ml. For example, if you purchased 100 µg of antibody, resuspend in 100 µl of pure distilled water. Dissolve the entire contents in water by mixing and lightly centrifuging at room temperature. It is possible to dissolve the contents in larger volume (200-500 µl) but the subsequent dilution may have to be adjusted. Insoluble lipoproteins may aggregate on storage. These do not interfere with the solubility of the antibody, spin down and use the clear supernatant.

How stable are the diluted antibodies?

Antibodies diluted as directed are very stable. Months and probably years. One study^Ref has sown extended shelf life for commercial reagents, beyond the manufacturer's expiration date. For antibodies used very diluted against low level antigens (<2K molecules/cell) is probably safer make fresh solutions monthly.

Do I need to freeze my antibody aliquots?

Each freezing cycle causes aggregation of the immunoglobulin and loss of titer. Avoid freezing aliquots that you expect to use out in the next couple of years, unless they are small aliquots which you will use completely within 1 or 2 experiments. You may freeze reference reagents in small aliquots for reference or reagents to be stored without preservative.

How long should I incubate the tissue/cells with the primary antibody?

Incubation for too short will not produce adequate signal. Incubation for too long can result in negative (unspecific) staining. Since the final result of your technique can only be determined at the end of your technique when no corrections can be made, the dilution and time of incubation for each antibody should be determined individually. In general, antibodies with known high affinity should be used at high dilution and overnight incubations. Antibodies with various affinities (polyclonal) must be experimented with to determine optimum time and dilution.

How should I fix my tissue/cells?

Please follow the link to our Fixation tips page.

Do I need to permeabilize the cells?

Upon fixation, all intracellular trafficking of molecules is stopped. Drying and lipid solvent treatments (acetone, ethanol, etc.) create massive holes in the greater sub/cellular structure. This allows antibodies to cross membranes (extracellular, nuclear, etc.) in fixed cells. There is no detergent that causes an antigen that was previously hidden (masked) to be exposed (unmasked). Low levels of detergents such as Tween 20 in the washing solutions reduce the surface tension and allow the tissues/cells to remain wet.

Should I use frozen or paraffin sections?

The most common histological preparative technique is formalin-fixed, paraffin embedded tissue. Some epitopes are more sensitive to fixation and embedding than others and can be masked (hidden) from the addition of affinity reagents. Various antigen retrieval methods exist to unmask a given epitope. These are not applicable to frozen sections. The optimal method will have to determined on a case by case basis. Crosslinking when fixating and embedding can prevent antigen degradation or physical relocation within the cell/tissue. It also eliminates bacterial contamination. Frozen sections can often lose morphological integrity, whereas paraffin embedded sections tend to retain it over multiple sections. Paraffin embedded sections will cause cell/tissue shrinkage that results in higher antigen density over a given section. Always be aware of the fact that your tissues/cells may contain various components that could interfere with your staining technique, such as endogenous enzymes (alkaline phosphatase, peroxidases), endogenous biotin and Fc receptors.

Immunohistochemistry (IHC) or immunofluorescence (IF/ICC)? Which is the most sensitive method?

IHC is relatively light-insensitive and allows the visualization of tissue architecture. Autofluorescence can sometimes make IF studies impossible. IF allows for staining of the same subcellular structure with different fluorophores.

Alkaline Phospatase (AP) or Peroxidase (HRP)? Which is the most sensitive?

NBT/BCIP substrate for AP is the most sensitive but, not widely used because the reaction is slow, does not allow adequate nuclear counterstain, the signal can diffuse and is not compatible with permanent mounting media. Development using DAB substrate for HRP is far more common due to speed of reaction, precise deposition and acceptable color contrast with nuclear stains.

Why do I need to use a positive control and a negative control?

A positive control is needed to monitor the test validity. A negative control is needed to establish a baseline or "zero", to prove that the test is the reason for the measured effect.