Typical Antibody Work-up:

In order to aid investigators in budgeting for antibody work-ups, we have prepared an outline of the typical sequence we follow when we work up a new antibody. This outline assumes that there is a commercially available antibody available that has been used for a similar application (tissue fixative, host range, etc.) as required by the investigator. Stages A, B, and C can usually be completed within 3 weeks. The time required to run the project tissue depends on the number of tissues and antibodies.

A. Identify potential antibody, the conditions for its use, and the appropriate controls:

Professional Time: 1 - 2 hours

B. Try antibody at recommended concentration with standard antigen retrievals:

We test 4 different antigen retrieval conditions using test antibody and the isotype, each with and without test tissue.

• No Ag Retrieval
• pH 6 Tris
• EDTA
• Proteinase K

Workprocess:

• Make control block
• Cut sections: 16
• IHC: 16
• Tech Time: 2 hours

C. Titer antibody:

In this step we test 4 different antigen concentrations, again using Ab and isotype, both with and without test tissue.

Workprocess:

• Cut sections: 16
• IHC: 16
• Tech Time: 2 hours

D. Test antibody on project tissue:

Finally, the actual project tissue is tested with the Ab and isotype (including controls of + or - test tisue).

Workprocess:

• Cut sections: 6
• IHC: 6
• Tech Time: 2 hours

E. Additional parameters that may require testing (not limited to the following):

• Blocking
• Detection systems
• Other antigen retrieval methods