Horseradish Peroxidase (HRP) Labeled Lectin Troubleshooting

ProblemCauseSolution
Weak or no Staining1. Low concentration of specific oligosaccharide on sample.
2. Low concentration of lectin conjugate.
3. Low concentration of Dye/Substrate.
4. Insufficient incubation time.
5. Inappropriate treatment of sample prior to labeling.
Causes #1 - #4
a. Increase incubation time.
b. Increase concentration of sample (on blot), lectin conjugate and/or Dye/Substrate.
a. Treat section or blot with a different blocking reagent.
High Background1. Lectin conjugate and/or Dye/Substrate is too concentrated.
2. Insufficient washing.
3. Insufficient blocking.
4. Sample contains enzymatic activity.
a. Decrease concentration of respective reagents.
b. Shorten incubation time.
a. Perform multiple washings and prolong washing time.
a. Treat section or blot with a different blocking reagent.
a. Determine if the sample contains activity which would give background staining in the absence of the Dye/Substrate.
Unexpected Staining PatternMultiple causesa. Perform control reactions.
b. Use other cytochemical techniques to prove or disprove finding.