Results:
Need description of the image as this: Observations about insulin granule distribution described as 1 text – see ru
ic
Need: figure legend for this pic. And also observations in the image. Properly presented fluorescence microscope images and observations from 1 selected fluorescently-stained microscope slide provided XXXXXXXXXXplease see ru
ic (need it detailed as its 3 marks)
Observations about stimulation conditions described in the text – please see ru
ic (need it detailed as its 3 marks)
Discussion:
Please see ru
ic and follow the each criteria asked and make sure each one is addressed- please see the marks to know how much to write for each.
GSIS
2.8mM glucose 16.7mM glucose 16.7mM glucose + X 16.7mM glucose + Y XXXXXXXXXX XXXXXXXXXX XXXXXXXXXX XXXXXXXXXX Conditions
[Insulin] ng/ml
Insulin ELISA Standards
12.8 6.4 3.2 1.6 0.8 0.4 0.2 0 XXXXXXXXXX XXXXXXXXXX XXXXXXXXXX XXXXXXXXXX 7.0000000000000021E-2 4.9000000000000009E-2 3.500000000000001E-2 0.05 [Insulin] ng/ml
Background Co
ected Abso
ance
Report assessment guidelines
The report has a three-page limit; any text exceeding this limit will not be marked. References
and Appendix are not included in this page limit. The minimum font size is 11. The minimum
margins are 2cm. A penalty of 20% will be incu
ed if these limits are exceeded.
The report should be written as a formal scientific communication. It is expected that the
eport will consist of the following sections:
1. Introduction and aims
2. Materials and methods
3. Results
4. Discussion and concluding remarks
The following sections are not included in the page limit:
5. References
6. Appendix – any information that you think should be included in the report can be
presented here and it can be refe
ed throughout the report.
All figures and tables should have an appropriate title and legend, placed either below or
above, respectively, with sufficient information for a reader to understand how the results
were obtained, including important information such as concentrations and duration of
stimulation conditions etc. If in doubt look at a peer reviewed article that has used a similar
experiment or assay for the best example.
Marking Scheme
Include your group’s student IDs in the file-name and front page of your report.
INTRODUCTION (/10)
Criteria Mark
What are β-cells? Why are they important? 2
How do β-cells function? What stimulates them and how can other substances
pertu
the normal responses to stimuli?
2
How does β-cell function change from healthy to insulin resistance and finally to
Type 2 Diabetes
2
Describe how you observed the function of β-cells in these practicals? Include
explaining the basic principal of a sandwich ELISA
2
Aims of the experiments 2
MATERIALS AND METHODS (/2)
Criteria Mark
Properly reference lab manual, and any variations to the protocol included 1
Co
ect formatting (third person, past tense) 1
RESULTS (/16)
Criteria Mark
Practical 1 data: Fluorescence microscopy images: properly presented in a
panel, including individual colour and
ight-field channels and an overlay of
lue and green colour channels, with appropriate labelling and scale bars
4
Practical 1 data: Observations about insulin granule distribution described as
text
1
Practical 1 data: Properly presented fluorescence microscope images and 4
observations from 3 1 selected fluorescently-stained microscope slide provided
Practical 3 data: ELISA standard curve and GSIS data presented as a column
graph with adequate figure title and legends
4
Practical 3 data: Observations about stimulation conditions described in the
text
3
DISCUSSION (/14)
Criteria Mark
Overview of the experiments. (What did you aim to do? What were your main
findings?)
4
Discuss the distribution of insulin staining in the β-cells and compare to
literature.
2
Discuss the differences between paired fluorescent microscopy samples.
*Discuss the visual observations from one provided microscopy slide
2
*Discuss how your ELISA results compare to the class average (class average 4
to be provided on Canvas)
Discuss what substances X and Y, from Practical 3, could be based on your
esults. Use references.
Concluding remark, summarise your findings and what was learnt. 2
REFERENCES (/2)
Criteria Mark
Five to ten journal articles used with consistent referencing style 2
Any referencing style is fine so long as it is consistent throughout the report (e.g. do not
efer to an article as [2] and then (Smith et al., 2009) elsewhere).
Sheet1
Average Stimulation Conditions [Insulin] ng/mL
2.8 mM glucose 0.9266
16.7 mM glucose 9.9448
16.7 mM glucose + X 18.2125
16.7 mM glucose + Y 1.4875
GSIS: TUESDAY Class Average
2.8 mM glucose 16.7 mM glucose 16.7 mM glucose + X 16.7 mM glucose + Y XXXXXXXXXX XXXXXXXXXX XXXXXXXXXX XXXXXXXXXX Conditions
[Insulin] ng/mL
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