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 ca
ied 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 mem
anes 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
emove 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 distu
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
oom 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
efore 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 co
ect 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 ca
ied out. Please have
this checked by your demonstrator before beginning your experiment.