BIOL2144 Cellular Communication Semester 1 2022

BIOL2144 Cellular Communication Semester 1 2022

Assessment: Lab report Practical 3

Immunofluorescence staining and tissue culture techniques

Assignment 10% of overall grade report is out of 40 marks

Include Name, Student number and Prac partner(s) name.

Please note you can find support materials at the end of this document for writing the Methods and Results sections of a report

Write a METHODS section for a biomedical paper based on the immunofluorescence staining activity in the prac notes. (20 marks total)

Why were the cells permeabilised and what was the name and type of reagent used? (2 marks)

What kind of molecule is Phalloidin? Describe its structure and how it interacts with its target? (2 marks)

What kind of compound is anti-Tubulin-FITC and how is it made? Where is the epitope for this reagent? (3 marks)

What is the difference between direct and indirect immunofluorescence? Which

of these techniques did you use to stain for tubulin and for vinculin in the prac? (3 marks)

Describe the cell staining in the wells that were stained in (F) staining- secondary antibody. Was there any green FITC staining? If so why? If not, why not? (2 marks)

RESULTS: Describe in words, your immunofluorescence staining activity along with your observations and image data from the prac. Include only 2 images in your report. Remember that a figure legend should be informative. (8 marks)

Background material

3. Immunofluorescence Example Methods section from BMC Cancer. 2017; 17: 672.

Experiments conducted in 2-dimensional cell culture, cells were seeded at ~50% confluence on glass cover slips. Following treatment, cells were fixed with ice cold 4% paraformaldyhide (PFA) pH 7.2 for 20 min. Cells were washed twice with phosphate buffered saline (PBS) then incubated for 1 h with primary antibody diluted in 0.25% bovine serum albumin (BSA) and 0.1% Saponin in PBS (BSP). After incubation with primary antibody, cells were washed twice with PBS and incubated with fluorescently conjugated secondary antibody diluted in BSP for 1 h. To visualize the cytoskeleton, cells were incubated with phalloidin diluted in BSP for 20 min. Cells were then washed three times in PBS and mounted using DAPI with Slow Fade Gold reagent. Images were taken using an Olympus UPlanFl 40X/0.75 objective on an Olympus BX50 microscope, utilizing a Roper Scientific Sensys Camera, and MetaMorph software.

Content and Writing Style of the Methods Section from RESPIRATORY CARE 2004 VOL 49 NO 10

Historically, the methods section was referred to as the materials and methods to emphasize the 2 distinct areas that must be addressed. Materials referred to what was examined (eg, humans, animals, tissue preparations) and also to the various treatments (eg, drugs, gases) and instruments (eg, ventilators) used in the study. Methods referred to how subjects or objects were manipulated to answer the experimental question, how measurements and calculations were made, and how the data were analyzed. The complexity of scientific inquiry necessitates that the writing of the methods be clear and orderly to avoid confusion and ambiguity. First, it is usually helpful to structure the methods section by:

1. Describing the materials used in the study

2. Explaining how the materials were prepared

3. Describing the research protocol

4. Explaining how measurements were made and what calculations were performed

5. Stating which statistical tests were done to analyse the data.

Second, the writing should be direct and precise and in the past tense. Compound sentence structures should be avoided, as well as descriptions of unimportant details. Once all elements of the methods section are written down during the initial draft, subsequent drafts should focus on how to present those elements as clearly and logically as possibly. In general, the description of preparations, measurements, and the protocol should be organized chronologically.

For clarity, when a large amount of detail must be presented, information should be presented in subsections according to topic. Within each section and subsection,

material should always be organized by topic from most to least important.

It must be written with enough information so that: (1) the experiment could be repeated by others to evaluate whether the results are reproducible, and (2) the audience can judge whether the results and conclusions are valid.

Writing a Results section

In the Results section, you present the data in a straightforward manner with no analysis of the reasons the results occurred or the biological meaning of the data (these comments are reserved for the Discussion). However, you should interpret the data, highlight

significant data and point out patterns, correlations, and generalizations that emerge. Also write this section using the past tense. Figures must be accompanied by a legend (below the figure).

A Results section that includes only a table or a figure and no text is not acceptable. Text must be given first, before tables and figures on a page if the tables and figures are included in the text. Unsummarised, or raw data should not be included. It is not appropriate to include redundant data and the same data should not be included in both table and figure form; rather, the data should be shown in the format that is most clear for the particular type of data collected and analyzed (see below).

The text of the Results section should describe the results presented in tables and figures

and call attention to significant data discussed later in the report. Do not repeat what is already clear to the reader from reviewing the tables and figures, which, if well constructed, will show both the results and experimental design.

A portion of the results text might read as follows:

The number of bacterial colonies increased up to 40C, but decreased at higher temperatures (Figure 1).The greatest amount of growth occurred between 35 and 40C.

In this example, Figure 1 refers to the graph in which the data are presented. In the same

sentence, the author says something about the data and refers the reader to the appropriate figure.

The figure (graph) may contain numerous data points (e.g., number of bacterial colonies at 1 C intervals from 0 to 60 C), but the author did not bore the reader with a description of each.Rather, generalizations are made concerning the relationships shown by the data, which the figure illustrates (a picture is worth a thousand words).