Optimization of Immunostaining techniques in Stenostomum virginianum


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Optimization of Immunostaining techniques in Stenostomum virginianum

  1. 1. Optimization of Immunostaining techniques in Stenostomum virginianum (Catenulida) Justin Waterfield Winthrop University Mentor: Julian Smith III
  2. 2. Phylogeny of Platyhelminthes <ul><li>Platyhelminthes is composed of two well-defined groups -Catenulida -Rhabditophora -originally three groups with the inclusion of Acoelomorpha </li></ul><ul><li>How the two groups fit properly under Platyhelminthes is still of debate </li></ul><ul><li>Stenostomum virginianum belongs to the group Catenulida </li></ul><ul><li>A possible synapomorphy for the two groups is the stem-cell (neoblast) system </li></ul>Smith et al (1986)
  3. 3. Neoblasts <ul><li>What do we know? -Differentiated cells in flatworms do not divide -Thus, neoblasts are the only dividing cells in flatworms (Egger et al 2007) </li></ul><ul><li>-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 </li></ul><ul><li>Study of the cell-cycle could answer whether or not the cells in the epidermis are proliferating cells </li></ul>Smith et al (1986)
  4. 4. Cell Cycle <ul><li>S-phase -replication of DNA takes place -takes 7.5-10hr -labeled with Anti-proliferating cell nuclear antigen (PCNA) </li></ul><ul><li>M-phase -Stop of cell growth and beginning of mitosis -shortest period of cell cycle, lasting only an hour -labeled with Anti-phosphoH3 </li></ul><ul><li>Proliferating cells would be marked in S-phase and M-phase </li></ul>From Ross & Pawlina, 2006
  5. 5. How does immunofluorescence work? <ul><li>Antibody “double-labeling technique” or the “sandwich” technique </li></ul><ul><li>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 </li></ul><ul><li>The primary anti-bodies used were anti-PCNA and anti-phosphoH3 </li></ul><ul><li>Fluorescent dyes that attached to the primary anti-bodies were Alexa flour 488/Cy2/Cy3/Cy5 </li></ul>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. 6. Dye list <ul><li>DAPI: Fluorescent dye that binds strongly to DNA. Colored Blue </li></ul><ul><li>Alexa Fluor 488/phalloidin: Fluorescent dye labeled protein, shows muscle fibers. Colored Green </li></ul><ul><li>Cy2: Fluorescent dye used to show PCNA in the Osmium/Formaldehyde fixations, shown as Green </li></ul><ul><li>Cy3: Fluorescent dye used to detect anti-phosphoH3 cells. Colored Red </li></ul><ul><li>Cy5: Fluorescent dye used to show PCNA in rest of the samples, shown as Yellow </li></ul>
  7. 7. Methods and Hypotheses <ul><li>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 </li></ul>Formaldehyde Osmium tetroxide
  8. 8. Methods and Hypotheses <ul><li>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 </li></ul>
  9. 9. Methods and Hypotheses <ul><li>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 </li></ul>
  10. 10. Osmium Fixed <ul><li>Osmium fixed worms produced a very bright mucus granule fluorescence </li></ul><ul><li>In this sample early formation of division planes can be seen </li></ul><ul><li>Image I shows DAPI/Cy2/Cy3 dyes </li></ul><ul><li>Image II shows DAPI/Cy2 </li></ul><ul><li>Lots of non-specific binding </li></ul><ul><li>Weak to no immunostaining </li></ul>Image I Image II
  11. 11. Acetone/formaldehyde Fixed <ul><li>In acetone fixation, mucus granule were not nearly as bright, allowed easier viewing of DAPI cells in the epidermis </li></ul><ul><li>Positive phosphoH3 cells can be seen as the red dots overlapping the DAPI cells </li></ul><ul><li>Images I & II show DAPI and Cy3 </li></ul>Image I Image II
  12. 12. Methacarn <ul><li>Protocol using Carnoy’s fluid in which methanol replaces the ethanol -Methanol -Acetic Acid -Chloroform </li></ul><ul><li>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 </li></ul><ul><li>Overall weak fluorescence in samples, although some phosphoH3 cells can be detected </li></ul><ul><li>No detection of PCNA </li></ul>Image I
  13. 13. 1-200 Dilution <ul><li>Acetone fixation </li></ul><ul><li>DAPI/Alexa 488phalloidin/AntiphosphoH3 Cy3 shown </li></ul><ul><li>Positive for phosphoH3 cells appear as the red dots </li></ul><ul><li>Alexa flour 488/phalloidin is used to illustrate the muscle fibers in the worm (green) </li></ul><ul><li>Image II shows a possible epidermal proliferating cell outside of the muscle fiber </li></ul>Image I Image II
  14. 14. 1-500 Dilution <ul><li>Acetone fixation </li></ul><ul><li>DAPI/Alexa 488phalloidin/AntiphosphoH3 Cy3 shown </li></ul><ul><li>Positive phosphoH3 cells in both images </li></ul><ul><li>Greater contrast at lower primary antibody concentration </li></ul><ul><li>Most phosphoH3 positive cells were located near the epidermis, with a few located outside of the muscle fibers </li></ul>Image I Image I Image II Image I
  15. 15. Overall Distribution <ul><li>In all of the samples, the highest concentration of DAPI positive cells were found along the body cavity and in division planes </li></ul><ul><li>No positive detection of PCNA cells in any of the samples </li></ul><ul><li>Scattered positive phosphoH3 cells throughout most of the samples </li></ul><ul><li>Most positive cells were found to be along the muscle fibers and bordering the parenchyma </li></ul>Image I
  16. 16. Conclusions <ul><li>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 </li></ul><ul><li>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 </li></ul><ul><li>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 </li></ul>
  17. 17. What next? <ul><li>Further research into techniques and protocols in electron microscopy </li></ul><ul><li>More data, more images, more samples </li></ul>
  18. 18. References <ul><li>Egger, Bernard, and S. Ishida. &quot;Chromosome fission or duplication in Macrostomum ligano (Macrostomorpha, Plathelminthes) - remarks on chromosome numbers in archoophoron turbellarians.&quot; (2004): 127-32. </li></ul><ul><li>Smith III, Julian P.S., and Sara Merlie. Stem Cells in Stenostomum virginianum. Department of Biology, Winthrop University. </li></ul><ul><li>Smith III, Julian P.S., Bernard Egger, Seth Tyler, Peter Ladurner, Johannes Achatz, and Sara Merlie. Neoblasts in Nemertodermatida. </li></ul><ul><li>Smith III, Julian P.S., Kevin Ryan, and Sara Merlie. Neoblasts in Catenulida. Department of Biology, Winthrop University. </li></ul>
  19. 19. Acknowledgements <ul><li>Winthrop University, for the opportunity and the resources provided to allow for undergraduate research </li></ul><ul><li>Dr. Julian Smith III, for without his guidance, knowledge, and especially patience, none of this would have been possible </li></ul><ul><li>All members of the Smith Lab </li></ul><ul><li>The support of my family, friends, and loved ones </li></ul>
  20. 20. Questions?