FAQ's
ELISA Troubleshooting Guide
Q: why my result is Poor on standard curve?
- Use appropriate diluent as blank. Make sure that the dilution is performed as according to protocol
- Briefly spin standard vial before opening. Make sure that there is no undissolved material after reconstituting
- Store standards as according to protocol
- Calibrate pipettes to make sure that the correct volume is dispensed
- Increase washing cycles
Q : why my result is Weak signal?
A : Use more antigens or antibodies for coating and use fresh substrate reagents.
Q: why my result is High background?
- Reduce the concentration of primary or secondary antibodies or use substrate with higher dilution
- Optimize incubation temperature for each experiment
- Calibrate pipettes to make sure that the correct volume is dispensed
Make sure that all reagents are mixed properly and equilibrated to room temperature before assay and make sure that the bottom of plate is clean
Q : why my result is No signal?
- Make sure that the instructions in the protocol is followed carefully
- Increase concentration of primary or secondary antibody
- Use fresh substrate reagents
- Check the settings (wavelength, filters, gain etc) of plate reader
Immunohistochemistry Troubleshooting Guide
Q: How do I dilute my antibody?
A: Here are the steps to dilute your antibody:
- Prepare a solution of the antibody in PBS or another buffer at the desired concentration.
- Determine the desired final concentration of the antibody in the sample. (For example, 1:50)
- Calculate the amount of antibody required for the desired final concentration. (ex. If the final concentration is 200ul, the antibody concentration should be 4ul)
- Calculate the amount of diluent (PBS or other buffer) required to achieve the desired final concentration. (ex. If the final concentration is 200ul, the diluent concentration should be 196ul)
- Carefully mix the antibody and diluent together in a sterile container.
- Mix the sample gently and thoroughly to ensure an even distribution of the antibody.
Q: At what temperature should I store my antibody?
A: Store at -20°C for long time (after 6 month), 4-8°C for short time.
Q: why my result is High background?
- Reduce the concentration of primary or secondary antibodies or use substrate with higher dilution
- Make sure that the fluorochrome does not overlap with one another.
- Make sure that there is no endogenous background caused by tissue itself. Check under fluorescence microscope prior to staining to identify autofluorescence.
Q: why my tissue is peeled off the slide?
A: Dry samples for 2-4 hours at 60°C. Tissue with high lipid content (eg breast tissues) should be dried for longer time.
Q : what is the resolution of the sectioning tissue?
A: Cut thinner slides for better resolution: 3-5 μm. Use a new/sharper blade.
Q: why my result of IHC is Low/No signal?
- Reduce fixation time if tissue is over fixation
- Increase fixation time or try other fixative if tissue is insufficient fixation
- Make sure that primary and secondary antibodies match one another.
- Use signal amplification methods (example: HRP Polymer)
- Make sure that paraffin is removed completely before staining.