Basis of the assay
- PR immunocytochemistry
- Dorsal raphe: E2 (Estrogen) induction of the PR is primarily an Estrogen-receptor-beta (ERb)-mediated event.
- Hippocampus: E2 up-regulates PR exclusively via the Estrogen-receptor-alpha (ERa).
- This differential regulation of PR can be utilized as a bioassay to test ER specificity of Selective estrogen receptor modulators (SERMs) in the Central Nervous system (CNS).
- ERb agonists (& antagonists)
- Timeline is more suitable for a secondary assay after initial screening of many compounds in mouse raphe (Taqman) Assay
- ERa antagonists
PR-ICC PD Model development takes 15 days.
- Collect and Section sample: Day 4-7
- ICC and Slide preparation: Day 8-13
- Analysis: Day 14-15
- vehicle (po)
- Estradiol (0.2 mpk, sc - E2 is not very orally bioavailable)
- Compounds (0.1 - 30 mpk, po)
PR Immunocytochemistry (ICC)
- Perfused tissue = better antigenicity
– acrolein/paraformaldehyde fixative
– brains sectioned on a vibrating microtome (40 um)
- Polyclonal or monoclonal antisera to the human PR (1:1000); recognize mouse, rat, human
- Biotinylated-secondary antibody (IgG)
- Avidin-biotinylated-HRPase complex (ABC)
– avidin-biotin binding is irreversible (high affinity)
– amplifies signal (avidin has 4 binding sites)
- Chromagen - diaminobenzidine (DAB)
– HRP + H202 -> DAB forms a brown precipitate
– Nickel sulfate - enhances signal - blue/black precipitate
- Reconstruct brain through raphe & hippocampus on slides & count the # or PR-ir cells under the microscope.
Critical steps in quantitative analysis
- The entity measured should be distinct from procedure used to identify it.
- The top layer of the section is utilized as a representative of the section.
- A distinct sub cellular region that needs to be observed.
- Consistency in tissue preparation, staining protocols and observation.
These results showed that it is possible to accurately complete a Immunohistochemical assay with human counting or automated computer analysis.