The overall goal of the following experiment is to use immunohistochemistry in order to visualize proteins of interest. For this example, IBA one for microglial and pan neuronal for neurons. This is achieved by first placing prepared sections into a blocking solution to prevent non-specific binding.
As a second step antibodies, which recognize the two different proteins of interest are placed on the sections overnight. Next sections are washed and incubated in secondary antibodies in order to add fluorescent tags to the primary antibodies. The dual labeling results can point out microglial stainings within fields of neurons.
Generally, individuals who are new to this technique will struggle at first because there are so many variables to adjust. Demonstrating this technique today will be Hazel May, an undergraduate in our laboratory. To begin carefully fix and section rodent brains as described in the accompanying text protocol, and then carefully store them at negative 80 degrees Celsius until they are needed.
When ready to continue with the protocol, remove the slides from the freezer and thaw them at room temperature. Once thawed, place the slides in a rack and wash them three times in PBS for five minutes each. From this point on, do not let the sections dry out.
Dry the edges of the slides and make a liquid repellent border around the edge of the slide with a pap pen to ensure an even covering of the liquid and reduce edge effects. Next block the sections by adding 300 microliters of serum in PBS to each slide. The serum should be from the species the secondary antibody is raised in.
In this case, donkey serum was used. Ensure the blocking solution extends to the edge of the slide and completely covers the tissue. To avoid uneven staining, place the slides into a humidity chamber to incubate at room temperature for one hour.
During incubation, prepare the primary antibodies dilute the IBA one antibody one to 5, 000, and the pan neuronal antibody one to 500 into a mixture of 1%donkey serum. In PBS, remove blocking solution and add 300 microliters of the antibody solution, which contains both antibodies of interest. Also, prepare three control slides.
Add 300 microliters of the IVA, one dilution alone to one slide, 300 microliters of the pan neuronal antibody dilution alone to a second slide, and 300 microliters of 1%donkey serum in PBS. On a third slide, place all the slides into a humidity chamber and incubate them overnight at four degrees Celsius the following morning. Wash the slides three times in PBS for five minutes each.
Prepare the fluorescent secondary antibody mixture by diluting both of them one to 250 in 1%Donkey serum in PBS. Add 300 microliters of the mixture onto each slide, and place the slides into a light tight humidity chamber for 60 minutes at room temperature. Then wash the slides three times in PBS for five minutes each, and finally, rinse them in double distilled water.
Protect the slides from light while washing. In PBS, add a few drops of an aqueous mounting medium that helps to preserve fluorescent signal intensity to each slide. Then cover, slip the slide and remove any remaining air bubbles using a cotton tipped applicator to seal the mounting media onto the slides and prevent the sections drying out.
Coat the edges with nail polish. The slides are left to dry flat for one hour in a light protected box before being stored in a light tight container wrapped in foil at four degrees centigrade. Once the nail polish has dried, bring the slides into the microscope room for imaging.
Prepare the room by turning off the overhead lights and blocking any other outside light sources. Turn on the microscope, the fluorescent light source and the imaging computer. Then place a slide on the microscope stage and bring it into focus.
Using the accompanying software, set the exposure for each wavelength separately. Avoid overexposing the sly to light for long periods of time to prevent photobleaching of the sample. Then acquire images in each channel without moving the section or adjusting the focus.
Combine the images using the imaging software and save them for later image analysis. Using the staining protocol just described, fluorescent images like the ones shown here can be easily obtained in the red channel IBA one positive microglia clearly appear, and in the green channel, pan neuronal positive neurons stand out above the background. The control images for both of these antibody stains appear.
Dark Double labeling with IBA one and pan neuronal marker shown here demonstrates mostly ramified microglia with long fine neuronal projections evident across cortical layers. Staining results like the ones shown here are also possible if the tissue quality is poor. Poor tissue quality is evidenced by the lack of clearly defined cell bodies and fragmented microglial and neuronal processes.
There is a lack of clarity of microglial staining and complete lack of neuronal staining, making the overlaid image not meaningful While attempting this technique. It's important to evenly cover the sections and remember not to let them dry out between the steps. After watching this video, you should have a good understanding of introductory immunohistochemical techniques.
