Optimization of Immunostaining techniques in Stenostomum virginianum

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Undergraduate research at Winthrop University

Undergraduate research at Winthrop University

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  • 1. Optimization of Immunostaining techniques in Stenostomum virginianum (Catenulida) Justin Waterfield Winthrop University Mentor: Julian Smith III
  • 2. Phylogeny of Platyhelminthes
    • Platyhelminthes is composed of two well-defined groups -Catenulida -Rhabditophora -originally three groups with the inclusion of Acoelomorpha
    • How the two groups fit properly under Platyhelminthes is still of debate
    • Stenostomum virginianum belongs to the group Catenulida
    • A possible synapomorphy for the two groups is the stem-cell (neoblast) system
    Smith et al (1986)
  • 3. Neoblasts
    • What do we know? -Differentiated cells in flatworms do not divide -Thus, neoblasts are the only dividing cells in flatworms (Egger et al 2007)
    • -Some Catenulids possess neoblasts in their epidermis, while no known Rhabditophora do -Is this a feature of Catenulids, or rather a primitive condition of flatworms? -S. virginianum along with other groups possess this feature, thus indicating a primitive condition of Platyhelminthes
    • Study of the cell-cycle could answer whether or not the cells in the epidermis are proliferating cells
    Smith et al (1986)
  • 4. Cell Cycle
    • S-phase -replication of DNA takes place -takes 7.5-10hr -labeled with Anti-proliferating cell nuclear antigen (PCNA)
    • M-phase -Stop of cell growth and beginning of mitosis -shortest period of cell cycle, lasting only an hour -labeled with Anti-phosphoH3
    • Proliferating cells would be marked in S-phase and M-phase
    From Ross & Pawlina, 2006
  • 5. How does immunofluorescence work?
    • Antibody “double-labeling technique” or the “sandwich” technique
    • The antibodies of the primary antibody host attach to the antigen of interest -Which is then rinsed -Placed into a solution of secondary antibody with dyes attached -The secondary anti-bodies are what contain the dye and allow fluorescence
    • The primary anti-bodies used were anti-PCNA and anti-phosphoH3
    • Fluorescent dyes that attached to the primary anti-bodies were Alexa flour 488/Cy2/Cy3/Cy5
    1° Ab binding: (mouse anti-PCNA / rabbit anti-phosphoH3) 2° Ab binding: (Dye-labeled donkey anti-rabbit / donkey anti-mouse) Wash away excess Ab
  • 6. Dye list
    • DAPI: Fluorescent dye that binds strongly to DNA. Colored Blue
    • Alexa Fluor 488/phalloidin: Fluorescent dye labeled protein, shows muscle fibers. Colored Green
    • Cy2: Fluorescent dye used to show PCNA in the Osmium/Formaldehyde fixations, shown as Green
    • Cy3: Fluorescent dye used to detect anti-phosphoH3 cells. Colored Red
    • Cy5: Fluorescent dye used to show PCNA in rest of the samples, shown as Yellow
  • 7. Methods and Hypotheses
    • Goal I: Osmium vs. Acetone/formaldehyde vs. Methacarn fixation methods -In a standard fixation the worms are placed into a vial filled with acetone after freezing on a LN2-cooled block - After an overnight storage in the freezer, warmed to room temperature and fixed with formaldehyde -In an osmium fixation the worms are placed into a vial filled with both acetone and 0.04% osmium - After an overnight storage in the freezer, warmed to room temperature, osmium worms already fixed -Test of Methacarn fixation, recommended DNA/PCNA protocol -Which one of these methods gives the best antibody staining and nuclear morphology? -I predict that the acetone/formaldehyde only fixations will be the more effective and optimal method of fixation for Stenostomum virginianum
    Formaldehyde Osmium tetroxide
  • 8. Methods and Hypotheses
    • Goal II: Optimization of primary antibody dilutions -Too high of a primary antibody concentration gives overstaining and high background; too low of a primary antibody concentration gives weak staining -I tested the effect of a 1:200 vs. 1:500 dilution of the primary antibodies -I predict that the 1-200 dilution will yield more positive proliferating cells -5uL of primary placed into 1mL buffer in 1:200 vs. 2.5mL in 1:500
  • 9. Methods and Hypotheses
    • Goal III: Overall distribution of PCNA/phosphoH3 positive cells -With the methods used above, what is the overall distribution of proliferating cells? -Where are they located? -Do the dyes exhibit non-specific binding? -S-phase lasts much longer than M-phase in the cell cycle -Therefore, I predict there will be more positive PCNA cells than phosphoH3 positive cells
  • 10. Osmium Fixed
    • Osmium fixed worms produced a very bright mucus granule fluorescence
    • In this sample early formation of division planes can be seen
    • Image I shows DAPI/Cy2/Cy3 dyes
    • Image II shows DAPI/Cy2
    • Lots of non-specific binding
    • Weak to no immunostaining
    Image I Image II
  • 11. Acetone/formaldehyde Fixed
    • In acetone fixation, mucus granule were not nearly as bright, allowed easier viewing of DAPI cells in the epidermis
    • Positive phosphoH3 cells can be seen as the red dots overlapping the DAPI cells
    • Images I & II show DAPI and Cy3
    Image I Image II
  • 12. Methacarn
    • Protocol using Carnoy’s fluid in which methanol replaces the ethanol -Methanol -Acetic Acid -Chloroform
    • Causes less shrinkage of the tissue, as well as preserves antigenic sites more effectively than Carnoy’s alone -Recommended by Sigma for Anti-PCNA antibody
    • Overall weak fluorescence in samples, although some phosphoH3 cells can be detected
    • No detection of PCNA
    Image I
  • 13. 1-200 Dilution
    • Acetone fixation
    • DAPI/Alexa 488phalloidin/AntiphosphoH3 Cy3 shown
    • Positive for phosphoH3 cells appear as the red dots
    • Alexa flour 488/phalloidin is used to illustrate the muscle fibers in the worm (green)
    • Image II shows a possible epidermal proliferating cell outside of the muscle fiber
    Image I Image II
  • 14. 1-500 Dilution
    • Acetone fixation
    • DAPI/Alexa 488phalloidin/AntiphosphoH3 Cy3 shown
    • Positive phosphoH3 cells in both images
    • Greater contrast at lower primary antibody concentration
    • Most phosphoH3 positive cells were located near the epidermis, with a few located outside of the muscle fibers
    Image I Image I Image II Image I
  • 15. Overall Distribution
    • In all of the samples, the highest concentration of DAPI positive cells were found along the body cavity and in division planes
    • No positive detection of PCNA cells in any of the samples
    • Scattered positive phosphoH3 cells throughout most of the samples
    • Most positive cells were found to be along the muscle fibers and bordering the parenchyma
    Image I
  • 16. Conclusions
    • Goal I: -In the Osmium fixations the mucus granules was highly fluorescent and could disrupt the ability to detect proliferating cells in the epithelium -The acetone fixations proved to have a significantly less non-specific labeling of the mucus granules allowing better distinction between the parenchyma and epithelial cells -Methacarn fixation proved to have poor immunostaining and imaging -The traditional method of acetone/formaldehyde is the optimal fixation method when using Stenostomum virginainum
    • Goal II: -The 1:200 vs. 1:500 dilutions showed no difference in immunolabeling between the samples -PhosphoH3 positive cells were found to be present in both dilutions, while there were no signs of any PCNA positive cells -From the samples collected there did appear however to be a sharper contrast in imaging from the 1:500 dilutions -Higher dilutions, such as 1:1000 seem plausible -Cost efficient
    • Goal III: -Distribution of cells are found to be highly concentrated along the formation of division planes, and all along body cavity -Some positive PhosphoH3 cells were found in a majority if the samples, however there was not a significant number found in the epidermis -A cell spends much longer (7.5hr-10hr) in S-phase than M-phase (1hr) -Expected to see lots of S-phase cells, but saw none -Does lack of PCNA detection indicate lack of PCNA in S. virginianum? - PCNA primary anti-body was raised in mouse anti-human, could be the result of ineffective binding -Cy3 will bind to the mucus granules as well as to some positive phosphoH3 cells
  • 17. What next?
    • Further research into techniques and protocols in electron microscopy
    • More data, more images, more samples
  • 18. References
    • Egger, Bernard, and S. Ishida. "Chromosome fission or duplication in Macrostomum ligano (Macrostomorpha, Plathelminthes) - remarks on chromosome numbers in archoophoron turbellarians." (2004): 127-32.
    • Smith III, Julian P.S., and Sara Merlie. Stem Cells in Stenostomum virginianum. Department of Biology, Winthrop University.
    • Smith III, Julian P.S., Bernard Egger, Seth Tyler, Peter Ladurner, Johannes Achatz, and Sara Merlie. Neoblasts in Nemertodermatida.
    • Smith III, Julian P.S., Kevin Ryan, and Sara Merlie. Neoblasts in Catenulida. Department of Biology, Winthrop University.
  • 19. Acknowledgements
    • Winthrop University, for the opportunity and the resources provided to allow for undergraduate research
    • Dr. Julian Smith III, for without his guidance, knowledge, and especially patience, none of this would have been possible
    • All members of the Smith Lab
    • The support of my family, friends, and loved ones
  • 20. Questions?