Fiber Type and Subcellular-Specific Analysis of Lipid Droplet Content in Skeletal Muscle

This protocol allows the quantification of lipid droplets and the analysis of their morphology and subcellular distribution in a fiber type specific manner. This technique enables the simultaneous processing of different muscles, saving time and avoiding artifacts that could occur when processed independently. It also permits the automatization of lipid droplets quantification.

Myosteatosis was assessed in marine muscles, but this protocol could be translated to humans in different conditions, such as obesity, aging, and cancer. Recognizing the same fibers in parallel section could be difficult, but remarkable structures such as axon tweaks or muscle spindles are good landmarks that will help the researcher locate the same fibers on both slides. To begin place two serial cryo frozen cross sections of the muscle specimen on two pre-labeled adhesion slides.

One slide to determine fiber types and the other to quantify lipid content. For immunohistochemical detection of muscle fiber type, surround the sections with an outline drawn with a hydrophobic pen. Place it in a humid chamber, and rinse with ice cold 0.1 molar PBS for one minute at room temperature.

Next, remove the PBS, add the blocking solution to the slide, and incubate for 90 minutes at 37 degrees Celsius. Later remove the blocking solution and incubate the slides for 90 minutes at 37 degrees Celsius with the solution containing the primary antibodies. Wash the slides thrice with PBS for five minutes each, at room temperature.

Add the solution containing the secondary antibodies and incubate the slides in the dark for one hour at room temperature, hereafter ensure to keep the slide away from the light and proceed with the three washes in PBS for five minutes each, then rinse the slide in double distilled water, after removing the excess water, mount the slide with antifade reagent and store it at four degrees Celsius protected from light. To stain the lipid droplet with bodipy, surround the muscle section with an outline drawn by a hydrophobic pen before rinsing it with ice cold 0.1 molar PBS for 10 minutes. Both slides must be stained simultaneously.

Fix the section with cold four percent paraformaldehyde without methanol for 10 minutes at room temperature. After first quick rinse wash the slides thrice with PBS for five minutes. Block the slide with 5%normal goat serum in PBS for one hour in human chamber.

Followed by incubating the slide with the solution of the primary antibody comprising 2%normal goat serum and rat anti-laminin in PBS for 90 minutes. After washing slides thrice in PBS, carry out the next incubation with the secondary antibody solution containing goat, anti-rat, Alexa fluor 647 antibody in PBS at room temperature for one hour. Make sure that the slide is protected from the light.

After incubation wash the slide thrice with PBS. Next, incubate the section for 20 minutes with dapi and bodipy solution. After a quick rinse, wash thrice with PBS, and once with double distilled water, then remove the excess water and mount the section with an anti-fade reagent.

Once the muscle is scanned, upload the captured digital images into any image processing software for reconstruction. Based on the fiber morphology and the histology of the muscle section, save the image in a suitable format with all the channels merged. For bodipy image observation and acquisition, set the parameters for a confocal microscope, with a 40 X oil immersion objective lens and a numerical aperture of 1.4, as described in the manuscript.

To avoid crosstalk between bodipy and 558, 568, and laminin Alexa fluor 647;Use the sequential scan mode on the confocal software. Excite bodipy and 493, 503 using the 488 nanometer laser line or argon laser line, and excite bodipy and 555, 568 using the 561 nanometer diode laser line. Finally, detect laminin Alexa fluor 647 with a 640 nanometer diode laser line, depending on the dye chosen, set the emission rates at 570 to 650 nanometers for bodipy and 493, 503.

And at 565 to 620 nanometers for bodipy and 558, 568. Set the emission range for laminin at 656 to 700 nanometers. Next, set the gain and digital gain appropriately to avoid detecting saturated pixels on the range indicator.

Correct the background signal by adjusting the offset. To identify the type of fiber on the confocal microscope, use a laptop on which the image of the previously reconstructed slide with the fiber type immuno detection can be checked. Once a group of fibers is correctly identified acquire the image with the bodipy and laminin channels.

Open each image with the aid of the bioformat's importer from Fiji. Under the view stack with option select hyper stack, then color mode and default. Make sure that the auto scale window is selected.

Use the free hand selection tool to manually select the sarcolemma of the fiber based on the laminin channel. And press T on the keyboard to record the region of interest on the ROI window. Go to Fiji's main window and click on analyze and set measurements.

Then on the popup window select area and for raised diameter, leave the remaining boxes unchecked and other parameters as they appear by default. Click on measure on the ROI window to obtain the area and minimal for raised diameter of the fiber selected and note them for later use. Calculate the value of one sixth of the minimal for raised diameter to delimit the central part of the fiber.

Click on the ROI window, Click on add to have a duplicate of the first ROI and select the second ROI that appears on the window. On Fiji's main window, click on edit, then selection and enlarge. To introduce the previously calculated value with a minus sign before the number and click on okay.

On the ROI window, click on add, A third ROI must appear, and delete to delete the second ROI. On the ROI window select both ROIs and click on more then ZOR and add, wait for a third ROI to appear which corresponds to the periphery of the fiber. If re-analysis of the same fibers is needed, save the ROIs by clicking on more then save.

Select the bodipy channel and open the threshold tool by clicking on image, adjust, and threshold tabs on Fiji's main window. On the threshold popup window set the values to 70 and 255. Then select YEN and black and white method.

Before clicking on dark background. Finally hit the apply tab. Go to Fiji's main window and click on analyze and set measurements.

On the popup window, select area, area fraction and limit to threshold. Leave the remaining boxes and parameters as default. Go to the ROI window and select the first ROI.

On Fiji's main window click on analyze, then analyze particles tool. On the analyzed particles window set the values from two to infinity and check the pixels box. Maintain the default circularity value and select summarize before clicking okay.

To obtain the values of the center and the periphery of the fiber, repeat the selection of the second and the third ROI each time. Then save the results by clicking on file, then save as in the summary window. Include the type of fiber on the name of the file.

This immunohistochemical protocol permits the recognition of mice, slow oxidative fibers, like type 1 and 2A, fast glycolytic fibers, such as type 2X and 2B, And the presence of hybrid fibers. By measuring the area and minimal for raised diameter of the muscle fiber a size dependent reduction was applied to obtain the central and the peripheral areas of each fiber. Remarkable structures of each muscle section, such as axon twigs or muscle spindles are good landmarks that could help locate the same fibers on both sides.

The settings described for acquiring bodipy laminin staining by confocal microscopy. Permitted, the visualization and analysis of the size, number, and distribution of lipid droplets from three to six fibers simultaneously. The method also allowed the quantification of three important parameters, namely the percentage of the fiber area occupied by the lipid droplets, the density, and the average size of lipid droplets.

Improper freezing procedure and failure to achieve the right temperature of isopentane resulted in an ice crystal formation inside the muscle fibers. When sections were not aligned transverse the lipid droplet quantification and comparison between fibers, muscles, or animals could not be performed. The processing of the second slide must be done simultaneously with the first.

Air drying the slide after cryosection for 15 minutes will significantly reduce the size and number of lipid droplets. Bodipy can be used to study the interaction of lipid droplets with other subcellular organelles, or protein stack with fluorescence of a different spectrum, or with genetically modified GFP models. Myosteatosis has been reported to be a poor prognostic factor for several diseases.

Therefore, this technique could be used to monitor the progression of a disease or to evaluate the effect of a treatment.