Detection of Amnionless Protein in

Kidney Sections

AuthorsNatasha TongeDr. Roberta Rivi

Acknowledgements

Dr. Roberta RiviDr. Sat Bhattacharya

Dr. Katia ManovaDr. Betul Altay-Ozer

Harlem Children SocietyElizabeth Lacy Lab

MSKCC

Wax in a Different Context

Tissue sample is embedded in paraffin wax

Wax in a Different Context

Tissue sample embedded in paraffin wax

Sliced with microtome

Wax in a Different Context

Tissue sample embedded in paraffin waxSliced with microtome

Placed in Tissue bath

Wax in a Different Context

Tissue sample embedded in paraffin waxSliced with microtomePlaced in Tissue bath

Mounted on a slide

Wax in a Different Context

Tissue sample embedded in paraffin waxSliced with microtomePlaced in Tissue bathMounted on a slide

Automatically Processed

http://mskweb3.mskcc.org/MolecularCytology/mccf-final.htm

Immunohistochemistry - a process used to detect proteins in a tissue section.

Antigen - a substance that provokes an immune response

Antibody – the protein complex that binds to antigens

Antibodies in a Different Context

Enzyme - A protein that speeds up a chemical reaction.

Substrate - The chemical that induces the chemical reaction

Immunohistochemistry - a process used to detect proteins in a tissue section.Antigen - a substance that provokes an immune responseAntibody – the protein complex that binds to antigens

Antibodies in a Different Context

primary antibody

secondary antibody

substrate

enzyme

Antigen

Enzyme - A protein that speeds up a chemical reaction.Substrate - The chemical that induces the chemical reaction

The Anatomy of the Kidney

Proximal Tubule - close to the glomerulus.

Ammnionless - protein located on the proximal tubule.

Cortex - outer region of the kidney. Most likely to find the glomerulus and proximal tubule here.Medula - inner region of the kidney. Lacks ammnionless.

http://upload.wikimedia.org/wikipedia/en/thumb/b/b0/Kidney_section.jpg/180px-Kidney_section.jpg altered by Natasha Tonge

The Kidney in Focus (more or less)

The Kidney in Focus (more or less)

The Past and Future of this Experiment

Tissue samples are sectioned, placed on slides and stained

The staining process, immunohistochemistry, makes use of antibodies to induce a color change

In future experiments the same process will be used topinpoint megalin and cubulin in abnormal mouse tissue. The results will then be accompanied by Western Blotting, Urine analysis and ELISA tests to characterize LRP Y2721-stop mice (mutants with shortened megalin sequences)

To Detect Amnionless in Kidney Sections:

That color change enabled us to see the location of the proximal tubules in the kidney sections.

Bibliography

"Human Hair." Wikipedia. http://en.wikipedia.org/wiki/Human_hair. 10 July 2006

Barnum, Susan R.. Biotechnology: An Introduction. 2. Belmont, CA: Thomson Brooks/Cole, 2005.

Campbell A., Neil, Jane B. Reece, Lawrence G. Mitchell, and Martha R. Taylor.Biology: Concepts &

Connections. 4th ed. San Fransisco: Benjamin Cummings, 2003.

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