Lab 5 - Fluorescence Guide F23 PDF

Final Lab: Fluorescence! Last time we discussed how protein expression can be detected via electrophoresis and western blotting. Today, we will discuss another technique that frequently uses antibodies to study the localization of proteins within a cell. Immunofluorescence is very common in Cell Bio...

Final Lab: Fluorescence! Last time we discussed how protein expression can be detected via electrophoresis and western blotting. Today, we will discuss another technique that frequently uses antibodies to study the localization of proteins within a cell. Immunofluorescence is very common in Cell Biology, as it provides useful (and beautiful!) data on where a particular protein is located. Today you will visualize tubulin and DNA in Ptk2 cells. The cells will be growing on glass coverslips in 6-well plates (just like in Lab 1!) You will use an antibody conjugated to the fluorochrome FITC to directly label tubulin. You will also use a fluorescent compound called Hoechst to directly label DNA. If you are lucky, you might even find some cells that are actively undergoing mitosis at the time of staining. FUN! Learning Outcomes: By the time this lab is complete, you should be able to: • • Describe how cells can be maintained in vitro. Describe how immunofluorescence can be used for the subcellular localization of proteins. BEFORE Lab 5, you must: • Carefully read the rest of this document and print it. • Complete a flowchart for Lab 5. Complete the pre-lab quiz on Brightspace. (Heads-up, the quiz includes questions from your previous labs!) • Assessments Related to the Lab: • Complete the In-Lab Worksheet (provided to you during the lab) and submit before you leave. McCarville, Garant and Tatar (2023/24) Antibodies… a quick review! Antibodies used in research applications typically belong to the IgG class and have a Y-shaped structure. The base of the Y is the constant domain, which is identical for all IgG-class antibodies from a single species. The branches of the Y are the variable regions, which complement the shape of the antigen and allow for binding to occur. This means that we can use antibodies to target virtually any protein (or other cell structure) that we wish to see! Indirect Visualization: Remember that it is common for research labs to make use of two antibodies: the “primary” and the “secondary” antibody. The primary antibody recognizes the protein of interest. The secondary antibody (raised in a different animal!) binds to the primary antibody and is attached to the reporter molecule. Direct Visualization: Similar to Lab 4, we will use a direct approach to label our protein – meaning that the antibody that recognizes the target protein is already conjugated to a reporter molecule. During Lab 4, when we were western blotting, we discussed how the enzyme HRP can be used as a reporter molecule to release light. In contrast, the reporter molecules used in today’s lab are fluorochromes. Fluorochromes: These molecules (also sometimes called fluorophores) emit light in the visible spectrum (i.e., colours!) There are many fluorochromes, each with a particular excitation and emission wavelength. Fluorochromes absorb light for an extremely short time and then re-emit light at a wavelength that is slightly shifted (~20-50 nm). The re-emitted light passes through a barrier filter in the microscope on its way from the stage to the eyepiece. This filter allows only light of the desired emitted wavelength to pass through it. FITC (Fluorescein isothiocyanate) is a fluorochrome with an excitation wavelength of approximately 495 nm and an emission wavelength of 517 nm (green light). Our tubulin antibody is attached to the reporter molecule FITC and is called Mouse anti-tubulin, conjugated to FITC. DNA can be labeled using a chemical compound called Hoechst. This fluorochrome binds directly to the DNA in the cell. When Hoechst is hit with ultraviolet light, it emits blue fluorescence at 460 to 490 nm. It is never conjugated to an antibody. It is important to appreciate that fluorochromes are found in every colour! For example, Cy3.5 (Cyanine 3.5) has an excitation wavelength of ~ 579 and an emission wavelength of 591 nm (red light). There are also purple ones, orange ones, etc. You just need to decide which colour you want (based on your microscope’s capabilities), and then you buy the appropriate antibody (or chemical reagent) that works with the immunofluorescence technique. For example, you could label the mitochondria by using a primary antibody that is specific to a subunit in ATP synthase (e.g. mouse anti-ATP synthase). Or, you could label microfilaments by using an antibody that will bind to actin. The possibilities are endless. Cell biologists customize their fluorochromes and antibody combinations to label different proteins or structures at the same time within the same cell. This allows them to see various components of the cell in contrasting colours so that a relative comparison (usually in location) can be made. McCarville, Garant and Tatar (2023/24) Fluorescence Protocol: Grow Cells: Approximately 24 hr prior to your lab time, we seeded Ptk2 cells into 6-well plates containing glass coverslips. By the time you arrive in the lab, the cells should be adhered to the top surface of the coverslip and have grown to our desired confluency. Fixing and Permeabilizing Cells: Each student will remove one coverslip from the 6-well plate and place it into a jar of ice-cold methanol for ~5 minutes. Hint #1: use the dissecting tool to help lift the coverslip out of the well. Hint #2: be hyper-aware of what side the cells are on; position the coverslip so that the cellside is facing the label on the front of the jar. Methanol fixes the cells so that the cell structures (e.g. the cytoskeleton) stay intact. It will also permeabilize the cell membrane so that the antibodies can enter the cell and bind their target protein. During the 5-min methanol incubation: • Prepare a humidity chamber by squirting water onto the filter paper in the glass Petri dish. The filter paper should be well dampened. • Remove the paper from the parafilm square and place the waxy square on top of the toothpicks in the humidity chamber. Your TA will walk around and pipette 45 µl of antibody (mouse anti-tubulin, conjugated to FITC) onto your piece of parafilm. • After 5 min, remove the coverslip and rinse it briefly by dipping it into a staining jar filled with PBS buffer. Antibody Incubation: • Place the coverslip cell side down onto the drop of antibody. Put the glass lid on the humidity chamber and wrap a piece of aluminum foil over the top to protect the fluorochrome from being quenched by the light in the room. Incubate the cells in antibody (i.e. leave the coverslip on the droplet) for 15 minutes. Noe: any used PBS can be poured down the blue sinks at your bench. • After 15 minutes, carefully lift the coverslip off the parafilm and place it into a staining jar containing fresh PBS for two minutes. Again, be careful to stay aware of what side of the coverslip your cells are on! This incubation will wash away un-bound antibody. • After 2 minutes, move the coverslip into a jar with fresh PBS for another 2-min wash. Do a 3rd wash with fresh PBS (i.e. total of three, 2-min washes). Pour used PBS down the blue sinks at your bench. DNA Labeling and Viewing Your Cells! • Bring your coverslip to the side bench and place it into a jar of Hoechst solution. Note: Hoechst is a potential mutagen and carcinogen (because it binds DNA) so wear gloves and be cautious. Leave your coverslip in the solution for 1 minute (watch the clock on the wall), then remove it and place it into a staining jar containing fresh PBS. Once your coverslip is in the jar of PBS, dispose of your gloves and put on a new pair! • At your bench, complete three more 2-min washes in fresh PBS. McCarville, Garant and Tatar (2023/24) • Retrieve a glass microscope slide from the box on your bench. Label one end with your initials and the date. Your TA will pipette a droplet (about 30 µl) of anti-fade mounting medium onto your slide. • When your final PBS rinse is complete, place your coverslip cell-side down onto the droplet of mounting medium. You are now ready to view the cells using the fluorescence microscope in the small darkroom next door! Either your Instructor or your TA will help with this part. 😊 We will use the UV filter to visualize the DNA. Once in focus, we will change the filter so that the FITC fluorochrome becomes visible! Before you leave the lab today: • • • Clean-up your bench. Make it look the same as when you arrived! Remove your gloves and wash your hands. Complete the In-Lab Worksheet and submit it before you leave. FYI, there are LOTS of other types of fluorescence-based techniques: Alternative fluorescent probes, such as GFP: In the description above, the cells were fixed (i.e. dead!) In some scenarios it is useful to detect protein localization in living cells. A common approach is to use gene editing to fuse the DNA that encodes for green fluorescent protein (GFP) to the cDNA that encodes for your protein of interest. The recombinant DNA is introduced into cells where it is expressed to produce a protein with a GFP tag. If the protein is exposed to blue light, GFP will glow green, providing valuable information regarding the location and function of proteins in living cells. Confocal Microscopy: The resolution in traditional fluorescence microscopy (as discussed above) is limited because you are looking at tissue with three dimensions from the top, and unfortunately, the whole area becomes illuminated, and the resulting image is a bit blurry. Confocal microscopy circumvents this problem by centering the light through a pinhole to focus on a single plane. When the ‘outof-focus’ light is blocked, the researcher can obtain a high-quality image that can be optically sectioned to provide further information. FRET: (Forster or Fluorescence Resonance Energy Transfer) uses fluorescence to measure interactions and distances between proteins. In this procedure, proteins are labeled with different fluorochromes. The key feature is that the emission wavelength of one fluorochrome overlaps with the excitation wavelength of the other fluorochrome. If the two proteins move into close proximity to each other, the energy from one fluorochrome is transferred to the other, and the emission from the second fluorochrome can be detected. Flow Cytometry: This technique is often used to gain information about a large population of cells. Flow cytometry passes the cells in a liquid suspension through a narrow channel. A laser scans the cells as they “flow” by, and based on light scatter, a computer generates information regarding the size and the shape of the cells, as well as detect the fluorescence emitted by any antibody-tagged proteins or other fluorochromes being used. Flow cytometry is commonly used to analyse apoptosis, detect proteins on the cell’s surface or intracellularly, or to determine which cells are in a particular stage of the cell cycle. If we consider the latter, when DNA is labeled with a fluorochrome (like Hoechst) the software can use the information regarding the amount of DNA in the nucleus to determine the phase of the cell cycle. For example, cells that have completed S-Phase (DNA replication) have twice as much DNA! Fluorescence-Activated Cell Sorting (FACS): This is a variation of flow cytometry where we isolate specific cells for future culturing and downstream applications. This time, as the cells are passed through the apparatus, they are separated into droplets - with only one cell per drop (amazing!). The instrument will monitor each cell’s information (i.e., size, shape, fluorescence, etc.) and can physically separate the cells into different containers for future use! FACS is commonly used to isolate a particular cell type from a heterogenous sample based on the expression of a cell surface marker. McCarville, Garant and Tatar (2023/24) Appendix I: How to Present Microscopy Images Often, microscopy images are presented as various panels within the same figure. An example is shown below. Note that in this figure, there is just one condition. If your experiment includes two different conditions (e.g. negative control and treatment images), they would be included as a second row within the same figure for easy comparison. Images provided by Dr. Katy Garant Figure Captions should go below the image(s). The following information should be included: • • • • • The specimen What protein / genetic material each colour is referring to Type of microscopy Total magnification used Any software used for imaging or analysis McCarville, Garant and Tatar (2023/24)