Antibody optimization

Antibody optimization is the process where antibody dilution, different pre-treatment method, incubation times, blocking and detection are fine tuned for the antibody in controlled conditions. Measurable values are antibody specificity, sensitivity, and stability.

Histola Research's antibody optimization services are flexible to meet clients needs. 

Please, do not hesitate to ask and discuss more about our optimization services!

        

 

 

Example of Histola's antibody and protocol optimization

 

Adopting an IHC protocol conventionally includes:

Antibody selection 
 
- On the basis of previous experiences and data sheets
- On the basis of a comprehensive literature search
- Uncommon antibodies almost always require a comprehensive literature search for the basis of pretreatment testing
  • Type of tissues chosen by the client
  • FFPE or frozen tissue sections
  • Determine the optimal control specimens:
    • Positive and negative control tissues
    • isotype controls 
  • Antigen unmasking:
    • HIER pH low
    • HIER pH neutral
    • HIER pH high
    • Proteolytic digestion
  • Different primary antibody dilutions and incubation conditions:
    • Fine tuning the optimal antibody dilution
    • Different incubation times and temperatures
  • Detection as client needs:
    • Tailored detection methods according to the client's needs
    • Polymer based amplification or ABC?
    • Chromogen or fluorochrome based visualization?
  • Counterstaining:
    • Usual hematoxylin, but many histological stains are possible use if needed
  • Small scale experiment on the client's samples 
    • Client selected samples and controls to determine possible variation in sample quality
  • Client approval
  • Client’s samples
    • After the client’s approval, the samples will be stained together with proper controls
  • Microscoping with digital camera and image analysis according to client needs
  • Reporting with conclusion