Practical class 1: Immuno-fluorescent staining of MIN6 beta-cells General Outline Today you will visualise the distribution of insulin granules in mouse pancreatic β-cells (MIN6) using...

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Practical class 1: Immuno-fluorescent staining of MIN6 beta-cells General Outline Today you will visualise the distribution of insulin granules in mouse pancreatic β-cells (MIN6) using immuno-fluorescent staining. Each group will have one glass coverslip on which cells have been settled and fixed onto. Following staining and mounting of the coverslip onto glass slides, each group will obtain images from two different regions for their reports using FLOID microscopes. Additionally, you will obtain multi-colour fluorescent images from 3 microscope slides, stained with 3 fluorophores, filling in the worksheet to describe features observed in each tissue or cell sample. Aims Students will be working in groups of four to: • Gain experience with immuno-fluorescent staining of mouse pancreatic β-cells • Gain experience using FLOID microscopes to visualize immuno-fluorescent staining • Learn the proper formatting of microscopy images • Present research data in a scientific report Flow chart Before class create a summary flow chart of the experiment to be carried out. Please have this checked by your demonstrator before beginning your experiment Protocol: Immuno-fluorescent staining Please ensure before you start you have the following: • One 3.5cm cell culture dish containing a glass coverslip with cells • Wash Buffer • Secondary antibody solution TO BE KEPT IN THE DARK • Mounting media containing DAPI TO BE KEPT IN THE DARK • Nail polish for sealing (see demonstrator for use) • One glass slide (YOU MUST CLEARLY LABEL WITH PENCIL): 1. Date 2. Group initials 3. Target of the primary stain (i.e. insulin) 4. Wavelength of the fluorophores used to stain (i.e. 488 for the 488ηm fluorophore) 2 Steps 1 - 7 have already been done for you. Please continue from step 8. 1. Wash: Remove cell culture media and wash twice with 500µL of PBS 2. Fix: Crosslink proteins to preserve cellular structures by using 500µL of 4% PFA for 20 mins 3. Wash: Remove PFA and wash twice with 500µL Wash Buffer (0.1% BSA in PBS with 0.01% Sodium Azide) 4. Permeabilise: After fixation, cell membranes are permeabilised by incubating the cells with 0.01% SDS for 5 minutes. This will allow the primary and secondary antibodies to access the intracellular space. 5. Block: Prevent non-specific staining by using 500µL of Blocking Buffer (DakoTM) for 1 hour 6. Remove blocking buffer, do not wash 7. Primary antibody incubation: Add 100µL pre-diluted guinea pig anti-insulin and incubate overnight at 4°C. Primary antibody has been removed from the cells and wash buffer has been added. 8. Wash coverslips 2 times: Gently remove the wash buffer from on top of the cells by SLOWLY tilting the culture dish 45° and using a p1000 pipette to SLOWLY aspirate (remove and discard) the liquid that has accumulated at the bottom of the well. Add 2mL wash buffer in the same manner by SLOWLY tilting the plate at 45° and SLOWLY adding it to the bottom of the well before SLOWLY setting it horizontally. Gently remove the wash buffer as described earlier. This counts as 2 wash steps. 9. Secondary antibody incubation: SLOWLY add 50µL of secondary antibody (prediluted anti-guinea pig Alexa Fluor 488) onto the coverslips from the corner of the glass as to not disturb the cells on the coverslip. SLOWLY place a square of parafilm over the secondary solution (the surface tension will hold the small amount of solution onto the coverslip). Cover the dish with aluminium foil, and incubate at room temperature for 1 hour in the dark. During the incubation time there will be demonstrations on how to image using the FLOID. FROM THIS POINT ON YOUR SAMPLES MUST BE KEPT IN THE DARK AS MUCH AS POSSIBLE! THIS IS TO PREVENT BLEACHING OF THE SECONDARY FLUOROPHORES 10. Wash coverslips 2 times: SLOWLY Remove parafilm from coverslip and wash as described in step 8, leave 1mL fresh wash buffer on the coverslip. A DEMONSTRATOR WILL COMPLETE STEPS 11 – 13 WITH YOUR GROUP 11. Dry coverslips: Carefully remove coverslips from the culture dish and place it (cell side up) onto paper towels. Let the coverslips dry for ~5 minutes at room temperature, keeping protected from light. 12. Mount coverslips: drop 30µL of mounting media containing DAPI (a blue fluorescent dye that binds to DNA, allowing visualisation of the nuclei of cells) onto each glass slide before carefully lowering the glass coverslip (cell side down) onto the droplet (aiming the center of the coverslip to the droplet). The weight of the coverslip and surface tension of the mounting media will draw out the mounting media to the edges of the coverslip, so it is not necessary to press down on the coverslip and squash the cells. Let the coverslip sit for at least 15mins in the dark before proceeding. If the coverslip is not fully on the glass slide very slowly push the edge back onto the glass slide. 3 13. Sealing coverslips: the coverslips need to be sealed with nail polish to prevent them moving around/off the glass slide whilst imaging and for longer term storage and potential future imaging. Imaging with the FLOID A demonstrator will run a mini lesson on how to obtain images during the 1 hour secondary antibody incubation time, please make sure you attend one of these before the end of the practical. When obtaining the images you must remember to: 1. Image representative cells: i.e. the cells that are an accurate depiction of all the other cells on the coverslip 2. Focus the image: you must adjust the focus once you’ve decided on which region to image so that it is not grainy 3. Put a scale bar: prior to saving your image you must select to show a scale bar, this is important for ensuring the cells that are imaged are of the correct size. E.g. a cell that may be abnormally large or small may have to be excluded from further analysis if they are not a representative cell 4. Save the images: to a USB and transfer them to your computer. Please check that you are able to open your images before you leave the class. Please use the supplied USB stick to transfer your images. Imaging Stained Pancreatic Tissue and Beta-cell Sections • Microscope slides are provided by the demonstrators during the practical session • Work with one slide at a time, as slides are shared between groups • For each slide, fill out the provided worksheet by recording slide information including Label, Tissue or Cell type, Structures observed at each fluorescent wavelength, and any key features of the slide. • Using the FLOID microscope, obtain representative images of each fluorophore and save images to USB (Remember your scale bar!) Worksheet (to be filled out with collected images) Section Label Tissue Cell type or Fluorescent wavelength Describe structure observed Key Visualised Features of Slide 4 405nm BLUE Nuclei 488nm GREEN 594nm RED 405nm BLUE Nuclei 488nm GREEN 594nm RED 405nm BLUE Nuclei 488nm GREEN 594nm RED 5 Practical Class 2: Glucose Stimulated Insulin Secretion assay and Insulin ELISA Day 1 General outline Today you will be doing a glucose stimulated insulin secretion (GSIS) assay on mouse pancreatic β-cells (MIN6 cells). As the name suggests you will simulate the MIN6 cells with low and high concentrations of glucose along with two unknown, insulin secretion modulators X and Y in combination with high glucose to trigger the secretion of insulin into the reaction media (KRBH). Substances X and Y have the ability to potentiate (increase) or supress (decrease) the amount of insulin secretion in the presence of high glucose concentrations. Figure 2. Six well cell culture plate containing MIN6 cells to be used for a GSIS. Each well contains MIN6 cells which will be stimulated as shown in the picture. Following the GSIS you will measure the amount of insulin that was secreted in each of the four conditions using an insulin ELISA and layout as shown in Figure 3 below. Figure 3. Insulin ELISA plate standard and sample loading order. Column 1 should contain the standards from low to high (top to bottom) and column 2 should contain the GSIS media samples in duplicates down the column. You will use two columns of the ELISA plate. One column for eight standards and the second column for the four stimulation conditions to be assayed in duplicate. At the end of next week’s practical you will be expected to determine which unknown substance is a potentiator Key: 2.8 mM glucose 16.7 mM glucose 16.7 mM glucose + X 16.7 mM glucose + Y 6 or suppressor and to postulate what either of these substances could be based on lecture content and the literature. Aims Students will be working in groups of four to: • Gain experience with handling mouse pancreatic β-cells • Perform glucose stimulated insulin secretion assays • Gain experience with insulin ELISA • Present research data in a scientific report Flow chart Before class create a summary flow chart of the experiment to be carried out. Please have this checked by your demonstrator before beginning your experiment.