Lab 3 Assignment_ Mounting and Fluorescent Microscopy

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Carleton University *

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3207

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Biology

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Apr 3, 2024

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Date:_______________ Name:_______________ Lab day:____________ Student #:_______________ Total: /83 NEUR 3207-Laboratory 3 Assignment: Mounting and Fluorescent Microscopy Instructions: Your lab assignment must be completed and submitted via Brightspace one week after your lab session. Your assignment will be graded along with your overall participation, safe lab practices (i.e., lab rules, and instructions), and cleanliness (cleaning workstation and equipment at the termination of the lab). Activity 1: Mounting and imaging GFP-positive mouse tissue sections (49 marks) 1) Indicate whether your group received vial A, vial B or vial C ( 1 mark ): _______________ 2) Insert your 2X image (merged), and 10X images (DAPI, GFP and merged) from the control tissue (i.e., no GFP expression) from the regions mentioned below. Make sure to include proper figure captions indicating the magnification, brain region and qualitative analysis of your image. Make sure your images are in a quartet, similarly to the image pasted below. Instructions titled NEUR3207 Lab 3 - Creating Figures Protocol , can be found on BrightSpace. ( 24 marks ) This is only an example image! a) Cortex (8 marks) b) Arcuate nucleus (8 marks) c) Ventral Tegmental Area (8 marks)

Date:_______________ Name:_______________ Lab day:____________ Student #:_______________ 3) Insert your 2X image (merged) and 10X images (DAPI, GFP, merged) for the GFP tissue that you mounted in the labs for the brain regions. Make sure to include proper figure captions, indicating the magnification, brain region and qualitative analysis for your image. ( 24 marks ) a) Cortex (8 marks) b) Arcuate nucleus (8 marks) c) Ventral Tegmental Area (8 marks) Activity 2: Microscopy and Theoretical Knowledge (19 marks) 7. Use all of the images (the ones you took and the ones emailed to you) to determine which transgenic mouse line you mounted tissue from. Justify your answer by comparing between the 3 transgenic mouse lines and the three different brain regions. Make sure to consider the presence or absence of fluorescence in select brain regions, as well as the pattern of expression distribution. Make sure to indicate which lines you received images from (i.e., Line A). (8 marks) 8. Explain how you were able to distinguish between the control tissue and the GFP expressing tissue? In your answer, reference what you saw on both the DAPI and GFP channels for both sets of tissue and what it means/why this is the case. (3 marks) 9. Explain how/why we are able to visualize GFP-positive neurons using the fluorescent microscope (hint: discuss excitation/emission wavelengths). (3 marks) 10. If one was to observe identical fluorescent patterns on all channels (i.e. DAPI, GFP, RFP) of the fluorescent microscope, what would this likely indicate? (2 marks) 11. Use your knowledge of transgenic mouse lines from the pre-lab reading to explain the following: A lab tech wishes to express green fluorescent protein (GFP) in vesicular GABA transporter (vGAT) neurons. What are two different ways that the tech could achieve this? Explain the process in detail and what happens when it comes to the expression of GFP. (3 marks) Activity 3: General Knowledge and Literature Analysis (15 marks) 12. Find a primary research article which examines the presence or function of NPY neurons within the cortex. Make sure to include the link to the article. Explain the following: a. What were the researchers investigating? ( 1 mark ) b. How did they research this? ( 2 marks ) c. What is their conclusion and future direction ( 2 marks ). 13. Find a primary research article which examines the presence or function of TH neurons within the ARC. Make sure to include the link to the article. Explain the following: a. What were the researchers investigating? ( 1 mark ) b. How did they research this? ( 2 marks ) c. What is their conclusion and future direction ( 2 marks ). 14. Find a primary research article which examines the presence or function of vGLUT2 neurons within the VTA. Make sure to include the link to the article. Explain the following:

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