The overall goal of this protocol is to use state-of-the-art immunohistochemistry to accurately and rapidly identify skeletal muscle fiber types. This method can help answer key questions in the field of muscle biology such as whether fiber type changes are precipitated by loss of functional proteins. Though this method can provide insight into many models of muscle disease, it can also be used in systems such as knock in and knock out models to reveal the greater impact of a single molecule on basic muscle function.
To begin, air dry frozen sections of mouse muscle on charged slides for about half an hour. Once dried, draw a border around the sections on each slide using a hydrophobic barrier PAP pen. Next, to block nonspecific antibody binding, load about 250 microliters of 5%BSA in PBS within the drawn borders and incubate the slides at room temperature for an hour.
Meanwhile, prepare a one to 50 dilution of the primary antibody in 5%BSA PBS. Make about 250 microliters per slide and store the antibody dilution on ice. After the block period, aspirate the block solution from the slides and add 250 microliter aliquots of the antibody dilution.
For a negative control, apply 5%BSA in PBS without primary antibody. Next, load the Petri dish with dampened filter paper and cover it in aluminum foil for an incubation chamber. Then incubate the slides at four degrees Celsius for 24 to 48 hours in the chamber or in a similar environment with the same humidity.
After the primary antibody incubation, aspirate the solution from the slides and wash the slides three times. For each wash, apply about 250 microliters of 5%BSA in PBS for about 10 minutes. While washing the slides, prepare one to 200 dilutions of the purified fluorophore conjugated secondary antibodies.
Prepare at least 250 microliters per slide and keep the solution in the dark and on ice. After the third wash, add 250 microliters of the secondary antibody dilution to the slides. Then incubate the slides for 90 minutes at room temperature in a dark humid environment.
After the incubation, aspirate the antibody solution and wash the slides three times as before. After washing away all of the excess secondary antibody, rinse the slides with PBS and allow them to air dry for 10 minutes. Lastly, mount the slides using a nonpermanent low viscosity aqueous mounting medium.
After the medium dries, seal the edges of the cover slip with nail polish and transfer the slides into an opaque storage box. Before imaging a slide, clean the cover slip with 70%ethanol. Then obtain digital images using an epifluorescence microscope.
Under low magnification, select a one square millimeter area of interest. To view Alexa 488 conjugated antibodies, use a 505 nanometer long pass filter. To view Alexa 594 conjugated antibodies, use a 595 nanometer long pass filter.
With the filters properly set, start collecting digital images. Be sure to include a scale bar for subsequent analysis. For the analysis, install Fiji in the macro provided with this publication.
Store the macro file in an easily accessed directory. Now, load a brightfield image of a selected section and navigate to the trainable weka segmentation option. In order to stipulate the cellular areas of the section, draw a line on a fiber and click add to class one.
Then to mark the extracellular domains, draw a line on the space between fibers and click add to class two. Repeat this process until five to 10 labels of each class have been defined. It can be difficult to mark the interstitial boundaries.
Using the zoom function allows one to mark these boundaries with greater precision. Next, select the train classifier option. On the resulting red and green overlay, manually add more labels to any parts of the image that were incorrectly segmented by the automated process.
Do this until the fibers marked in red are appropriately separated from the intervening spaces marked in green. Then click on get probability and save the image in a new file folder. Now, repeat the entire process using the fluorescent image taken from the same field.
Label the immunostained fibers as class one and all the nonfluorescent regions as class two. Ultimately, save the resulting probability map to the same folder where the processed brightfield image was stored. Now, open one of the previously saved probability maps in Fiji.
Draw a straight line on the scale bar provided by the imaging software. Select set scale and input the correct values based on the scale bar. Then toggle the global option to standardize the scale to every image.
Now, run the Tyagi macro for fiber quantification and the navigation pane will appear. Then open the images host folder. In the associated dialogue box, change the background dropdown value to light.
The set measurements function can be used to define which parameters should be measured. CSA and feet diameter are the most pertinent measures to quantifying fiber morphology. After running the macro, the folder will then be populated with images of the fiber outlines and spreadsheets with the measurements made by the poor analysis function.
Tibialis anterior and soleus muscles were analyzed using the described methods because they are mostly composed of either fast twitch or slow twitch fibers. Several different myosin heavy chains were identified using specific antibodies. A substantial fraction of the soleus muscle fibers expressed type one and type two A myosin heavy chains and a fair amount of the remaining fibers were positive for type two X.By contrast, virtually no type two B fibers were evident.
By comparison, stained tibialis anterior muscles were composed mostly of type two B and type two X myosin heavy chain fibers with smaller contributions from fibers expressing type two A chains and virtually no fibers expressed in type one chains. Morphometric analysis of a tibialis anterior muscle probed with SC-71 antibodies directed to type two A myosin heavy chain provided several image transformations used to make probability maps. From this easy automated analysis, the data proved fully comparable to data generated using much more tedious manual calculations.
All together, the analysis yield the data from about 6, 000 total individual fibers. The high data throughput made the statistical analysis far much more feasible. After watching this video, you should have a good understanding of how to immunostain muscle sections and use the provided semi-automated software to facilitate analysis of muscle fiber type and composition.
While attempting this procedure, it is important to remember that muscle can be regionally heterogeneous and that analysis of many muscle fields is required for rigorous assessment of muscle fiber type and composition. In combination with this procedure, other methods like Trichome staining can be performed in order to answer additional questions like whether fibrosis develops in parallel with changes in fiber type composition.
