Biology (Neuroscience)

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LAB5_6ExamplesHowtowritethemethodssectionofaresearchpaper.docx

Bio310

How to write the methods section of a research paper.

Kallet RH & Fikret Erdemir

The methods section of a research paper provides the information by which a study's validity is judged. Therefore, it requires a clear and precise description of how an experiment was done, and the rationale for why specific experimental procedures were chosen. The methods section should describe what was done to answer the research question, describe how it was done, justify the experimental design, and explain how the results were analyzed. Scientific writing is direct and orderly. Therefore, the methods section structure should: describe the materials used in the study, explain how the materials were prepared for the study, describe the research protocol, explain how measurements were made and what calculations were performed, and state which statistical tests were done to analyze the data. Once all elements of the methods section are written, subsequent drafts should focus on how to present those elements as clearly and logically as possibly. 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 sub-sections according to topic. Material in each section should be organized by topic from most to least important. It is generally recommended that the materials and methods should be written in the past tense, either in active or passive voice.

It is generally known that the material and methods section is a relatively easy section of an article to write. Therefore, it is often a good idea to begin by writing the materials and methods section, which is also a crucial part of an article. Because “reproducible results” are very important in science, a detailed account of the study should be given in this section. If the authors provide sufficient detail, other scientists can repeat their experiments to verify their findings.

Example from scientific literature:

Saul A. Villeda et al, Nat Med. 2014 June ; 20(6): 659–663. doi:10.1038/nm.3569.

Immunohistochemistry

Tissue processing and immunohistochemistry were performed on free-floating sections following standard published techniques30. Mice were anesthetized with chloral hydrate (Sigma-Aldrich) and transcardially perfused with 0.9% saline, and brains were removed and fixed in phosphate-buffered 4% paraformaldehyde for 48 h before cryprotection with 30% sucrose. Free-floating coronal sections (40 μm) were incubated overnight with rabbit anti-Egr1 (1:500, 588, Santa Cruz), rabbit anti–c-Fos (1:500, 4-17, Millipore) or rabbit anti-pCreb (1:1,000, Ser133, 06-519, Millipore) primary antibodies, and staining was revealed using biotinylated secondary antibodies and the ABC kit (Vector) with diaminobenzidine (DAB; Sigma-Aldrich). Individual cell numbers were quantified by Egr1 and c-Fos, and pCreb was quantified as the mean signal intensity using NIH ImageJ software.

Example from Bio310 UHart:

Comparative Distribution of Thalamic Glial Cells using GFAP Marker through Immunohistochemistry

Materials

Mice brain tissue was collected in the following manner. Avertin (0.011-0.014 mL/g or 2.5% working sol) was used to anesthetize mice, which were then suffused with 0.9% saline solution and then a 4% Paraformaldehyde (PFA) solution (Lab manual, 20). The whole brains were then postfixed at 4 ̊C overnight in 4% PFA (Lab manual, 20). After 24 hours had elapsed, the brains were washed thoroughly in Phosphate Buffered Saline (PBS) at room temperature, with 1 brief wash, and 3 washes for 25 minutes each. Brains were sliced at 50 μM thickness, collected on slides, and were frozen at -20 ̊C.

Solutions that were used included PBS, Block solution, I ̊ mouse monoclonal antibody solution of Anti-Glial Fibrillary Acidic Protein (GFAP), II ̊ antibody solution of anti-mouse GFAP, 4',6-Diamidino-2-Phenylindole (DAPI) nuclear counterstain, and Semi-permanent mounting medium. PBS was a mixture of 0.01 g NaCl/mL of dissolved oxygen, 0.2 g KCl with pH solution to pH 7.4, 1.44 g of Na2HPO4 brought to l liter with distilled water, and 0.24 g KH2PO4 (Lab manual, 21). Block solution was a mixture of PBS, 0.5% Triton X-100, and 5% serum (Lab manual, 22). Glass slides and cover slips, cuvettes, and micropipettes were used to mount the brain samples.

Methods

Primary antibody was added to two slides of the mouse brain section. 400 μL of total solution (1:400 concentration of I ̊ antibody) was placed on each of the two slides and was left overnight (Immunohistochemistry Principle, 13). Two slides with 8 sections of whole brain each were carefully removed of their cover slips with tweezers and were briefly rinsed with PBS for one minute at room temperature, and were then washed with PBS twice more for 10 minutes each. During the washing, the secondary antibody was prepared in the following manner. The II ̊ antibody solution of anti-mouse GFAP was concentrated 1:1000 (Immunohistochemistry Principle, 13).

After the final wash in PBS, the slides were removed from the PBS, the back of the slides were dried with a kimtech wipe, and the slides were placed flat in a slide holder. 250 μL of the II ̊ antibody was pipetted onto each slide from the made solution, and the slide holder cover was placed over the slides to produce a dark environment. Each section of the brain was covered with the antibody solution. The lights in the lab were also dimmed to minimize light interference with the auto-fluorescent dye in the II ̊ antibody solution. The slides remained in the dark box for an hour before they were washed in PBS once again for 20 minutes at room temperature. The slides were removed from the PBS, the back of the slides were dried with a kimtech wipe, and the slides were placed flat in the slide holder before 1000 μL of DAPI was pipetted on each slide. The DAPI was placed over each brain section and the slides were covered and kept in the box for 10 minutes. The slides were then washed in PBS briefly and then once again for 10 minutes. The instructor allowed the slides to dry in the dark for 5 minutes and a cover slip was mounted on top with AquaPoly mount. The slides were then dried in the dark at room temperature for 15 minutes, and were stored in the dark at 4 ̊C overnight.