oringham's blog

In the Spring of 2018, the Methods project was conducted as part of the coursework for the Writing in Biology class taught by Professor S. Brewer. The goal of the Methods Project is to explore the importance of explicit, detailed and concise writing when composing a scientific paper. Scientific writing must reflect these qualities in order for replication of experiments and analyses to take place by other interested scientists. The methods project demonstrates how omission of important information can lead to large differences in results of an experiment.

In Spring of 2018, as part of the Writing in Biology Course at the University of Massachusetts Amherst, the Methods project was completed in order to enhance students’ understanding in how to create effective written pieces in the biological sciences. The original and replicate figures created of Calliandra haematocephala possess qualitative differences despite having the same methods followed in order to create them. Figure 1A and 2A are photos of different blooms on the tree, resulting in a large visual difference in the figure. Figures 1B and 2B are of the trunk and branches of the tree, and appear to be the most alike between all three components of the figure with only very slight differences in branches captured. Figures 1C and 2C also contain distinct differences. The borderlines for the states within the United States of America are outlined, and Florida is filled in red in figure 1C. Figure 2C does not possess borderlines for the states of the United States, and the state of Florida and the United States is a gray color. Additionally, different fonts were used to label each figure, but possess the same qualities of capitalization and bolded text.

Overall, this laboratory exercise demonstrates the importance of the use of a primary antibody to gain specific and localized staining of a particular cellular structure when using indirect immunofluorescence. Additionally, the use of direct cellular targets to label specific cellular components is also shown to be useful, especially when comparing cellular structures across cell types.

The fluorescence of control LLC-Pk1 cells was distributed indiscriminately throughout the cellular components due to the absence of primary antibody for the fluorescently tagged secondary antibody to bind to. The lack of affinity for a specific target within or around the cell allowed for the fluorescently tagged secondary antibody to freely bind to any structure or surface that it desired. This resulted in a fluorescent image in which most cellular structures and area surrounding the cells was tagged with fluorescent dye, ultimately brightening much of the area under the cover slip and outputting a low fluorescence intensity in the area of interest (tubulin). The fluorescence of the indirect immunofluorescence stained cells localized specifically to tubulin structures due to the presence of a primary antibody which targeted antigens located on tubulin molecules within the cells. This allowed for multiple secondary antibodies (which possess a high binding affinity for the primary antibodies) to bind to the primary antibodies, achieving a highly amplified fluorescence intensity on only tubulin structures. This is visually represented in Figures 1 and 2, and quantitatively measured in Table 1. Direct targeting of the actin cytoskeleton in LLC-Pk1 cells and NIH 3T3 cells allowed for clear visual distinctions to be seen in the structural composition of the actin cytoskeleton in each cell type. The actin cytoskeleton of the NIH 3T3 cell group is considerably more spread out within the cell, rather than localized to one region of the cell. This is due to the large role that fibroblasts play in maintaining structural integrity of connective tissues in living organisms. These cells must possess qualities of strength and durability throughout the cell, which is provided by the widely distributed actin cytoskeleton. Additionally, fibroblasts are able to migrate as individual cells, so the actin cytoskeleton extending within the branched structure could aid mobility on the cellular level. This structure can be visualized in Figures 3 and 4. Conversely, the actin cytoskeleton of the epithelial cells is more localized around the nucleus of the cell, rather than spread throughout the cell. This can be explained by the epithelial cells inability to migrate as individual cells, so a widely distributed actin cytoskeleton is not necessary. Additionally, the localization of the actin cytoskeleton specifically around the nucleus results from the large role that the actin cytoskeleton plays in cell division by transporting cellular components and preparing the cell to divide. This localization can be seen in Figures 3 and 4.

Fluorescent stained phalloidin was used to directly label the actin cytoskeleton of both fibroblasts (NIH 3T3) and LLC-Pk1 cells. Images of each cell type were taken at 10X and 100X magnification under a fluorescent filter, and in phase contrast. In visually examining the images, it is clear there is a stark difference in actin cytoskeleton composition for each cell type. Figures 3B and 4B demonstrate the actin cytoskeleton of LLC-Pk1 cells as a localized group of filaments around the nucleus and more compact. Figures 3D and 4D depict the cytoskeleton of NIH 3T3 cells under fluorescence, which appear to extend away from the nucleus and are less compact. 

Images of pig kidney epithelial (LLC-Pk1) cells that were treated with both primary (anti-tubulin) and secondary (goat anti-rat FITC) antibodies for indirect immunofluorescence were taken at 10X magnification (Figure 1B & 1D) and 100X magnification (Figure 2B & 2D) under a fluorescent filter. Additional images were taken in phase contrast at 10X and 100X magnification (Figure 1A & 1C, Figure 2A & 2C, respectively). Additional LLC-Pk1 cells were treated in the absence of a primary antibody, (with buffer solution and secondary antibody) to create a control group for indirect immunofluorescence. These cells were imaged at 10X magnification (Figure 1C & 1D) and 100X magnification (Figure 2C & 2D) under a fluorescent filter. It can be visually discerned that there is a large difference in localization of fluorescent dye and ability to visualize tubulin structures between the two conditions. Figure 1B demonstrates a clear localization of fluorophores to the cells, specifically of tubulin structures around the nucleus. Figure 1D shows contrasting visual data, where fluorophores are seen indiscriminately binding to cellular structures as well as resting on parts of the slide that contain no cells. These differences are again highlighted, showing the direct localization of dye to microtubules, and random dying of miscellaneous cellular structures at a higher magnification in Figures 2B and 2D, respectively. Additionally, quantification of fluorescence was measured for both control and indirect immunofluorescence groups for all magnifications. It is apparent that the control group possesses a lower fluorescence intensity in localized tubulin areas than the group using the primary antibody for indirect immunofluorescence at 10X magnification (22.123, 46.028, respectively) and 100X magnification (31.249, 63.962, respectively) (Table 1).

Indirect immunofluorescence staining and the direct labeling of components within the cell are two ways in which one can visualize parts of the cell that are otherwise too microscopic or transparent to identify. In this lab, we explore how primary antibodies impact efficacy of targeted cellular structure staining with respect to indirect immunofluorescence. Additionally, phalloidin coupled with fluorescent dye is used to directly label the actin cytoskeleton of two different cell types in order to visualize and investigate the organizational differences between the two cell types’ actin cytoskeleton structure.

A possible explanation for the discrepancies between figures 1A and 2A could be that the replicated photo was taken of a different bloom on the tree. This could have happened because there was a lack of explicit detail explaining exactly which bloom was photographed in the original figure. Additionally, the angle was not specified in the methods whether is was to the right or left side of the ceiling of the conservatory, which could have helped in taking a more exact photo of the bloom. Figures 1B and 2B do not appear to be much different from one another. There are slight discrepancies between the amount of leaves captured at the top of the tree, but many of the factors that could have resulted in major differences in the figure were controlled by the limited angle options due to the corner placement of the tree. Differences between figures 1C and 2C can be explained by the insufficient explanation of how to create state borders on the figure map website. The default world map setting does not include state borders, so a box must be checked in order for the borders to be seen. With this box checked, each individual state can be highlighted and Florida can be colored in.These directions were not explicit in the methods section, and this step was missed in the execution of the map making for the replicate figure. Additionally, the type of font was not detailed in the methods, resulting in a different font used to label the figures on the replicate.  

Part A both figures holds the widest discrepancy between the two figures. Figure 1A shows a flower with a bloom pointed at a 45° angle with respect to the ceiling of the conservatory. Figure 2A depicts a bloom at a 90° angle with respect to the ceiling of the conservatory. Part B of both figures displays a little discrepancy between both figures. Part C both figures varies slightly with respect to the outlining of the states within the United States, and the highlighted portion of florida. Figure 1C displays a map in which the states of the United States are outlined with borderlines, and the state of Florida is highlighted in red. Figure 2C does not contain state borders within the United States, and the state of Florida is colored grey along with the rest of the United States. Additionally, differences in font are noted for the figure labels in each figure.

The goal of the Methods Project is to explore the importance of explicit, detailed and concise writing when composing a scientific paper. Scientific writing must reflect these qualities in order for replication of experiments and analyses to take place by other interested scientists. The methods project demonstrates how the smallest omission of important information can lead to large differences in results of an experiment. The figure created for the methods project details Calliandra haematocephala, the Powder Puff Tree, and its indigenous locations throughout the world. The exhibit of the Powder Puff Tree at the University of Massachusetts is very easy to spot, and contains only one tree with a very small number of blooms. For these reasons, this tree was believed to be a one of the best options on campus to complete this project with accurate replication. The limited number of blooms offered little room to choose an incorrect flower, and the corner placement of the tree in the conservatory made it very simple to photograph the tree from the same angle. Additionally, the limited amount of countries the tree is found to be indigenous in limits the amount of error in missing a country on the map portion of the figure. Controlling for these factors allows for a more accurate and analogous replication for the figure of Calliandra haematocephala.