Trouble 1: Weak signals
1. Not enough antigens. Antigens may be lost after se tion preparation. Examine the fixative or fixation procedure.
2. Low antibody concentration. Examine the antibody concentration.
3. Antigen masked. Liberate antigen by antigen retrieval procedures or agents.
4. Blocking too strong. Strong blocking may reduce the signal. Examine the blocking condition and the species blocking agent.
5. Too much washing. Examine the time and number of washings and the composition of washing solution especially detergent concentration.
Trouble 2: High background or appearance of nonspecific staining
1. Antibody concentration too high. Excess antibody can enhance non-specific signal. Examine the antibody concentration.
2. Endogenous peroxidase activity remaining. When using peroxidase for chromogenic reaction, endogenous peroxidase enhances nonspecific staining. Use longer denaturing time or strengthen the denaturing condition.
3. Not enough blocking. Some antigen and antibody have preference of blocking agents, change the blocking agents or check the blocking conditions.
4. Not enough washing. Increase the number or time of washing.
5. Too long incubation with antibody. Reduce the antibody concentration or shorten the incubation time.
To be used for research only. DO NOT use for human gene therapy or clinical diagnosis.