A. Clarify the source and quality of antibody. Study the characteristics of the antigen. ***Check the literature.***

1. Quality of the Antibody

a. Polyclonal or monoclonal?

b. Purification method if polyclonal?

c. Specificity tested?
d. Raised in which animal?

2. Characterize the antigen

a. Intracellular or extracellular?

b. Which species produced the protein?

c. Size?

B. Start with immunofluorescence.

1. Check specimen for autofluorescence
2. Common fluorescent dyes

a. Fluorescein-isothiocyaniate (FITC)

b. Tetramethylrhodamine (TRITC)

3. Test this method on frozen or lightly paraformaldehyde-fixed samples.

C. Test EM fixatives at the light level with immunoperoxidase or immunogold methods.

1. Common methods

a. Peroxidase-anti peroxidase (PAP)

b. Avidin-biotin complex (ABC)

2. Various fixatives

a. Glutaraldehyde

b. Paraformaldehyde

c. Glutaraldehyde/paraformaldehyde

d. Periodate-lysine-paraformaldehyde (PLP)

3. Reaction products are visible at both LM and EM.

D. Select EM protocol if necessary (see Appendix D)