In the TDP43-localization experiment, you will use commercially available antibodies to determine the location of TDP43 in CAD cells. The ability to bind specific proteins using antibodies, or immunoglobulins, is critical in immuno-fluorescence labeling analysis. Antibodies are typically 'raised' in mammalian hosts. Most commonly mice, rabbits, and goats are used, but antibodies can also be raised in sheep, chickens, rats, and even humans. The protein used to raise an antibody is called the antigen and the portion of the antigen that is recognized by an antibody is called the epitope. Some antibodies are monoclonal, or more appropriately “monospecific,” and recognize one epitope, while other antibodies, called polyclonal antibodies, are in fact antibody pools that recognize multiple epitopes. Antibodies can be raised not only to detect specific amino acid sequences, but also post-translational modifications and/or secondary structure. Therefore, antibodies can be used to distinguish between modified (for example, phosphorylated or glycoslyated proteins) and unmodified protein.
Monoclonal antibodies overcome many limitations of polyclonal pools in that they are specific to a particular epitope and can be produced in unlimited quantities. However, more time is required to establish these antibody-producing cells, called hybridomas, and it is a more expensive endeavor. In this process, normal antibody-producing B cells are fused with immortalized B cells, derived from myelomas, by chemical treatment with a limited efficiency. To select only heterogeneously fused cells, the cultures are maintained in medium in which myeloma cells alone cannot survive (often HAT medium). Normal B cells will naturally die over time with no intervention, so ultimately only the fused cells, called hybridomas, remain. A fused cell with two nuclei can be resolved into a stable cell line after mitosis.
Generating monoclonal antibodies.
To raise polyclonal antibodies, the antigen of interest is first purified and then injected into an animal. To elicit and enhance the animal’s immunogenic response, the antigen is often injected multiple times over several weeks in the presence of an immune-boosting compound called adjuvant. After some time, usually 4 to 8 weeks, samples of the animal’s blood are collected and the cellular fraction is removed by centrifugation. What is left, called the serum, can then be tested in the lab for the presence of specific antibodies. Even the very best antisera have no more than 10% of their antibodies directed against a particular antigen. The quality of any antiserum is judged by the purity (that it has few other antibodies), the specificity (that it recognizes the antigen and not other spurious proteins) and the concentration (sometimes called titer). Animals with strong responses to an antigen can be boosted with the antigen and then bled many times, so large volumes of antisera can be produced. However animals have limited life-spans and even the largest volumes of antiserum will eventually run out, requiring a new animal. The purity, specificity and titer of the new antiserum will likely differ from those of the first batch. High titer antisera against bacterial and viral proteins can be particularly precious since these antibodies are difficult to raise; most animals have seen these immunogens before and therefore don’t mount a major immune response when immunized. Antibodies against toxic proteins are also challenging to produce if they make the animals sick.
Generating polyclonal antibodies.
In your experiment, you will use a primary antibody to bind TDP43. Then a secondary antibody will be used that is specific to the conserved region of the primary antibody. The use of secondary antibodies allows researchers to tag the primary antibody. In our assay, the tag is a 488 nm fluorescent dye that will enable us to visualize the location of TDP43 in CAD cells using microscopy.
Part 1: Treat cells for TDP43-localization experiment
The cells you split in the previous laboratory session were used to prepare the plates you will use today for the TDP43-localization experiment. First, 25,000 cells were seeded in each well of the 12-well plate and the cells were incubated for 24 h. Then the small molecules were added at final concentrations of 3 μM or 30 μM in serum-free media according to the plate map shown below. The cells were incubated with the small molecule compounds in the 37 °C incubator for 1 hr prior to the start of our laboratory session today.
Though the small molecule compounds were already added to your cells, it is good practice to calculate the dilutions that will were used for your treatment conditions. All of the ligands were prepared at two stock concentrations for this experiment, which will allow you to test each ligand at two treatment conditions. Use the following information (and the table below) to calculate the volume of each component should be added for each condition:
- Small molecule stock concentrations = 1 mM and 10 mM
- You will use the 1 mM stock to prepare the 3 μM experimental treatment condition and the 10 mM stock to prepare the 30 μM treatment condition.
- To account for possible effects of DMSO (the small molecules were dissolved in DMSO), the volume of DMSO added to the control should be equivalent to the volume of small molecule added for the experimental treatments.
- 1 mL of total volume per well
|| small molecule (stock concentration = 1 mM)
|| small molecule (stock concentration = 10 mM)
| DMSO negative control
| small molecule treatment concentration = 3 μM
| small molecule treatment concentration = 30 μM
To visualize the localization of TDP43 via antibody staining, the treated CAD cells must be fixed. By fixing the cells, all cellular processes are stopped and cells are adhered to the coverslips. Though the Instructors completed this, the protocol is included below for you to review.
- Following the 1 hr incubation, aspirate the liquid from the well and immediately add 400 μL of 4% paraformaldehyde to fix the cells.
- Incubate at room temperature for 10 min.
- Collect the 4% paraformaldehyde in the correct waste stream using a P1000 pipet.
- Wash with 500 μL of 1X PBS.
- Add 1X PBS then remove using a P1000 pipet. Collect the PBS in the correct waste stream.
- Complete a total of 2 times. Leaving 1 mL of 1X PBS on the cells in the final wash.
- Leave all wells with 1 mL 1X PBS, parafilm the sides and move the 12-well plate into the 4 °C cooler.
In your laboratory notebook, complete the following:
- Calculate the volumes of DMSO and / or ligand that were added to each condition (you can include the above table).
Part 2: Perform antibody staining for TDP43-localization experiment
Complete primary staining steps
To ensure the steps included below are clear, please watch the video tutorial linked here: [Staining].
Immunofluorescence staining chamber
- Obtain a 12-well plate seeded with CAD cells from the front laboratory bench.
- Gather an aliquot of 1 X TBS from the front laboratory bench.
- Prepare 1.2 mL solution of 0.2% Triton X-100 (v/v) (starting concentration of Triton = 10%) in 1X TBS in a micro centrifuge tube. This will be used to permeabilize the cells in Step #6.
- Prepare 2.5 mL solution of 1% BSA (v/v) (starting concentration of BSA = 10%) in 1X TBS in 15ml conical tube. This will be used to prepare the primary antibody solution in Step #8.
- Obtain a staining chamber from the front bench and add a damp paper towel to each side of the parafilm. Label parafilm with experimental details.
- Obtain a pair of tweezers from the front laboratory bench.
- Carefully use the tweezers to lift the coverslip from the 12-well plate.
- Practice plates with coverslips will be available at the front laboratory bench.
- When you are confident with your ability to retrieve the coverslips from the wells, move one coverslip from each condition from your 12-well plates to the staining chamber. Cell-side UP! The cell-side of the coverslip is the side that was facing up in the well of the 12-well plate.
- Quickly permeabilize the cells by adding 150 μL of the 0.2% Triton X-100/TBS solution to each coverslip and incubate for 10 min at room temperature.
- Aspirate the 0.2% Triton X-100/TBS solution and add 150 μL of BSA blocking solution to each coverslip, then incubate for 60 min at room temperature.
- With 15 min remaining of the blocking solution incubation, prepare the primary antibody.
- Dilute the rabbit anti-TDP43 antibody 1:1000 in the 1.2 mL aliquot of BSA blocking solution.
- Aspirate the block solution and add 150 μL of the diluted primary antibody solution to each coverslip before moving the next. Do not let the coverslips dry!
- Carefully move your staining chamber to the 4 °C cooler.
- Incubate samples at 4 °C in the primary antibody solution for 1 h.
Complete secondary staining steps
- Retrieve the staining chamber with your coverslips from the 4 °C cooler.
- Wash each coverslip by pipetting 200uL of TBS-Triton to the top of the coverslip, then use pipet to remove liquid.
- Complete a total of 3 washes. At the final wash leave the liquid on the coverslip.
- Retrieve aliquot of diluted secondary antibody, Alexa Fluor 488 goat anti-rabbit (1:200 in blocking solution) from front bench.
- Aspirate the wash liquid from one coverslip and immediately add 150 μL of the diluted secondary antibody to the coverslip.
- Complete this step for each coverslip individually as it is important that the coverslips do not dry!
- Cover your coverslips to protect them from light.
- Incubate samples at 4 °C in the secondary antibody solution for ~1 h.
- Make sure to have TBS solution available before you start. Aspirate the secondary antibody solution off the coverslip and immediately add 150 μL of TBS. Do not let the coverslips dry out during this process.
- To complete the post secondary wash, add 150 μL of TBS per coverslip, let incubate at room temperature for 3 min covered, then aspirate.
- To add DAPI, dilute the DAPI stain 1:1000 in TBS and add 150 μL DAPI per coverslip. Let incubate at room temperature for 10 min covered, then aspirate.
- Add TBS as in step 2 for the final wash and leave for 3 min. Do not aspirate.
- Obtain glass slides from the front laboratory bench and label your slides with all of your experimental information and group name, add one drop (20 uL) of mounting media to the slide.
- Aspirate the final TBS wash and using tweezers place the coverslip cell-side down on the mounting media "spot" on the microscope slide. Try your best to avoid bubbles by slowly placing the coverslip over the mounting media.
- The cell-side of the coverslip is the side that was facing up in the staining chamber.
- Complete Steps #5-6 for coverslips from all of the coverslips you stained.
- Add one small drop of nail polish to each side of your coverslip to seal it to the glass slide.
In your laboratory notebook, complete the following:
- Why are the cells permeabilized prior to staining?
- Why is it important to wash the secondary antibody from the coverslip before imaging?
- What stain is used following secondary antibody? What cellular component is stained in this step? And why is this useful?
Part 3: Get a start on your homework
During the incubation times, prepare the methods revision due on M1D8!
Using feedback to improve your writing is an important step to developing your scientific communication skills. For this homework, work with your laboratory partner to incorporate the feedback you received to revise your methods draft. In addition, include the protocols you used to evaluate purified TDP43 and for the aggregation assay on M1D4 and M1D5.
See the description on the Homework tab for more information.
- DMSO (from Sigma)
- small molecules (from ChemBridge)
- permeabilization buffer: 0.2% Triton in Tris buffer saline (TBS) (from Invitrogen)
- blocking buffer: 1% bovine serum albumin (BSA) in TBS (BSA from Sigma)
- 1:1000 primary antibody to TDP43, rabbit (from Proteintech)
- 1:200 Alexa Fluor 488 goat anti-rabbit IgG (from ThermoFisher)
- 1:1000 DAPI (from ThermoFisher)
- Fluoromount G (from Southern Biotech)
Next day: Image TDP43-localization experiment and complete data analysis
Previous day: Learn best practices for mammalian cell culture and seed CAD cells for TDP43-localization experiment