Fluorescent Labeled Lectin Troubleshooting

ProblemCauseSolution
Weak or no Staining1. Low concentration of specific oligosaccharide on sample.
2. Low concentration of lectin conjugate.
3. Insufficient incubation time.
4. Photobleaching
Causes #1 - #3
a. Increase incubation time.
b. Increase concentration of conjugate.
a. Avoid exposure to light.
High Background1. Lectin conjugate is too concentrated.
2. Insufficient washing.
3. Autofluorescent sample.
a. Decrease concentration of respective reagents.
b. Shorten incubation time.
a. Perform multiple washings and increase washing time.
a. Perform multiple washings and prolong washing time.
a. Use fluorochrome with different excitation and emission spectrum.
b. Use a different lectin conjugate (enzyme or colloidal gold).
Unexpected Staining PatternMultiple causesa. Perform control reactions.
b. Use other cytochemical techniques to prove or disprove finding.