Optimization of Immunostaining techniques in Stenostomum virginianum (Catenulida)

Justin WaterfieldWinthrop University

Mentor: Julian Smith III

Phyloge ny o f Platyhe lminthe s

• 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)

Ne oblas ts

• 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)

Ce ll Cyc le

• 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 Ro s s & Pawlina, 2006

How do e s immuno fluo re s c e nc e work?

1 ° Ab binding:(mous e anti-PCNA / rabbit anti-

pho sphoH3)

2° Ab binding:(Dye -labe le d donke y anti-rabbit / donke y anti-mous e )

Wash away e xc e s s Ab

•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

Dye lis t

• 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

Me thods and Hypo the s e s

• 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

Formalde hyde Osmium te troxide

Me thods and Hypo the s e s

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

Me thods and Hypo the s e s

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

Osmium Fixe d

• 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

Ac e tone /fo rmalde hyde Fixe d

• 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

Me thacarn

•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

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

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 IImage I

Image II

Image I

Ove rall Dis tribution• 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

Conc lus ions

• 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

What ne xt?

• Further research into techniques and protocols in electron microscopy

• More data, more images, more samples

Re fe re nc e s

• 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.

Ac knowle dge me nts

• 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

Que s tions ?