You will use commercially available antibodies to identify γH2AX foci in your experiment. 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 the γH2AX foci. 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 double-strand breaks via microscopy. As a reminder, during the last laboratory session MEF cells were treated with H2O2 +/- As for the γH2AX assay. Today we will discuss how to permeabilize the cells, which will enable the antibodies to enter the cells and bind γH2AX.
Part 1: Perform antibody staining for γH2AX assay
Complete primary staining steps
Immunofluorescence staining chamber
- Obtain your 12-well plates 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) in 1X TBS in a micro centrifuge tube using the 10% Triton stock is at the front laboratory bench.
- Prepare 2.5 mL solution of 1% BSA (v/v) in 1X TBS in 15 mL conical tube. 10% BSA stock is at the front bench.
- One of the preparations will be the blocking solution used in Step #8 and the other preparation will be used in Step #9 for the primary antibody solution.
- Obtain a staining chamber from the front bench and add a damp paper towel to each side of the parafilm.
- Label the parafilm with experimental details.
- Obtain a fine gauge (26 3/8) needle and a pair of tweezers from the front laboratory bench.
- Carefully press the tip of the needle against the benchtop to bend it into a right angle such that the beveled side of the needle is the interior angle.
- Use the 'hook' created with the needle to lift the coverslip from the bottom of the well, then use the tweezers to 'catch' the coverslip.
- 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 mouse anti-γH2AX 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!
- Cover your staining chamber with the lid to incubate at room temperature.
- Incubate samples in the primary antibody solution for ~1 h.
Complete secondary staining steps
- Aspirate the primary antibody and wash each coverslip by pipetting 200uL of TBS to the top of the coverslip, then use pipet or aspirator 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-mouse (diluted 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.
- 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 (15 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 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 2: Image γH2AX experiment
Diagram of a fluorescence microscope.
As discussed in prelab, two antibodies were used in the H2AX assay. The first antibody, or primary antibody, was anti-γH2AX and raised in a mouse. The secondary antibody was anti-mouse and raised in a goat, more importantly, this molecule is conjugated to a fluorescent dye tag called Alexa Fluor 488. The Alexa Fluor 488 tag is a bright, green fluorescent dye that is excited at 488 nm. To visualize the abundance of double-strand breaks in your H2AX assay samples, we will use fluorescence microscopy.
In fluorescence microscopy the specimen is illuminated with a wavelength of light specific to the excitation of the fluorescent tag used to target the feature of interest. The excitation wavelength is absorbed by the fluorescent tag, which causes it to emit light at a longer, less energetic wavelength. Typically, fluorescence microscopes used in biology are an epifluorescence type with a single light path (the objective) for excitation and emission detection, as depicted in the diagram above.
Fluorescence, or epifluorescence, microscopes are composed of a light source, an excitation filter, a dichroic mirror, and an emission filter. The filters and the dichroic mirror are specific to the spectral excitation and emission characteristics of the fluorescent tag. To visualize fluorescence, light at the excitation wavelength is focused on the sample. The light emission from the sample is focused by the objective to a detector.
Due to timing reasons, the Instructors will complete the imaging for the coverslips prepared during this laboratory session. To ensure you are familiar with the imaging procedure, each team will see a demonstration provided by the Instructor.
In your laboratory notebook, complete the following:
- What is the type of microscope used?
- What is the light source used by this microscope?
- Which objective are you using to image the γH2AX data?
- What is a benefit of using a higher objective for this imaging? A consequence?
- What is a benefit of using a lower objective for this imaging? A consequence?
- Where do you expect to see the 488 nm signal in your images? What might it mean if you don't see the signal where it is expected? What might it mean if you see the signal where it is not expected?
- 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 γH2AX, mouse (from Millipore)
- 1:200 Alexa Fluor 488 goat anti-mouse IgG (from ThermoFisher)
- 1:1000 DAPI (from ThermoFisher)
- Fluoromount G (from Southern Biotech)
Next day: Complete data analysis for γH2AX experiment
Previous day: Prepare and treat cells for γH2AX experiment