11-Fluorescence_microscopy_Report_2023 (1)

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Jan 9, 2024

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LIFE 212 Laboratory Report 11 Name: ___________________________ Due___________________________ Section: ___________________________ Fluorescence Microscopy Lab 50 pts total plus Notebook Conclusion (4 pts) 1. List the name of each fluorophore and the cellular structure(s) to which it bound. (6 pts) 2. An early step of the immunofluorescence protocol required use of a solution containing TBS (Tris-buffered saline) and the blocking agents 1% BSA and 2% goat pre-immune serum. Explain the purpose of the BSA and goat serum in this step. What could happen if you did not use them? Keep in mind that the 2 o Antibody-fluorophore conjugate was raised in goat. (4 pts: 2 points each) 3. The Lab 11 procedure referred to three important controls that should be performed to ensure the appropriateness of each reagent for our experimental goals and detection system. One of the controls required incubating the fixed/permeabilized cells on the cover slip with the 2 o Ab-Fluorophore conjugate alone (and leaving out the primary (1 o ) antibody). If fluorescent features were visible using the secondary antibody (2 o ) alone, the protocol is 1

LIFE 212 Laboratory Report 11 Name: ___________________________ Due___________________________ Section: ___________________________ not optimized and other reagents should be tried. What could have occurred if fluorescent features are visible when using the secondary antibody (2 o ) alone ? (3 pts) 4. What is the purpose of using calcium and magnesium in the PBS wash solution? (3 pts) 5. Referring to Lab 11 Section IV A and Figure 2, assume that you are studying 2 proteins to see if they co-localize in the same subcellular compartment. You immuno-labeled each one of the proteins and used 2 o antibodies conjugated to fluorophores with the following excitation and emission wavelengths: Probe 1: excitation 452 nm emission 558 nm Probe 2: excitation 440 nm emission 560 nm a) If the epi-fluorescence filter blocks in the microscope are specific to the above listed excitation and emission spectra, will you be able to use both probes simultaneously in the same cells to differentiate two different cell components? Will you be able to visually distinguish between the two stains? Explain. Draw a diagram to illustrate your explanation (6 points: 3 points for the diagram; 3 points for the explanation) b) What kind of control experiment would you design to test for the suitability of these probes to simultaneously detect and differentiate two different cellular proteins? (4 points) 2

LIFE 212 Laboratory Report 11 Name: ___________________________ Due___________________________ Section: ___________________________ 6. Import your photos into Powerpoint. Assemble them into a single 4 part figure. Use this space to tape color printout(s) of your 4 photomicrographs (1 image per fluor and one merged image). (10 pts) 3

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LIFE 212 Laboratory Report 11 Name: ___________________________ Due___________________________ Section: ___________________________ 7. Calculate the final magnification of the photo above by measuring the diameter of a single cell (measure from one edge of the cell to the other), where final magnification = printed size / true size. Use an estimate of 15  m for the true length of 3T3 fibroblasts. Show your work. (6 pts) 8. Write a single four-part figure legend for your 4 photos. Be sure to include the final magnification calculated in the question above and label the photograph A, B, C, D to refer to each photo. (Look up examples online of peer-reviewed articles to see the specific types of additional information to include in a micrograph figure legend.) (8 pts) Notebook Conclusion (4pts) The conclusion should be a concisely written summary of the experimental results using technical language. Describe each objective from your Introduction individually and frame in context with the experimental results. Vague and/or incomplete conclusions will not receive any credit. 4

LIFE 212 Laboratory Report 11 Name: ___________________________ Due___________________________ Section: ___________________________ 5

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