Imaging. Data Mining. Writing. Devices.

 

Converting a qualitative Progesterone receptor (PR) Assay to a quantitative assay

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

2013-02-08-BrainAtlas

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

Groups:(n=4-6 mice/group)

    1. vehicle (po)
    2. Estradiol (0.2 mpk, sc - E2 is not very orally bioavailable)
    3. Compounds (0.1 - 30 mpk, po)

2013-02-08-Timeline

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.

 

2013-02-08-Staining

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.

 

2013-02-08-graph

2013-02-08-graphs

These results showed that it is possible to accurately complete a Immunohistochemical assay with human counting or automated computer analysis.