Fiber Type Identification of Human Skeletal Muscle

Skeletal muscle is a heterogeneous tissue, with distinct cellular metabolic and contractile properties that are dependent on whether the cell (fiber) is slow twitch (type I) or fast twitch (type II). The fiber type can be identified by examining the myosin heavy chain (MHC) isoforms, which differ from each other in several ways, including contraction time, speed of shortening, and fatigue resistance¹. Major MHC isoforms include type I, type IIa, type IIb, and type IIx and their metabolic profiles are either oxidative (type I and IIa) or glycolytic (IIx, IIb)¹. The proportion of these fiber types varies in muscle type and between species. Type IIb is widely found in rodent muscle. Human muscles do not contain any type IIb fibers and consist predominantly of MHC isoforms type I and IIa fibers, with a small proportion of IIx fibers². Protein expression profiles vary between different fiber types and can be altered with aging³, exercise^4,5, and disease⁶.

Measuring cellular responses in different skeletal muscle fiber types is often overlooked or not possible due to the examination of muscle homogenates (a mix of all fiber types). Single-fiber western blot allows for the investigation of multiple proteins in individual muscle fibers⁷. This methodology has previously been utilized to produce novel and informative single-fiber characteristics that were not possible to obtain using homogenate preparations. However, there are some limitations of the original single-fiber western blot methodology, including the time-consuming nature, the inability to generate sample replicates, and the use of expensive, sensitive enhanced chemiluminescence (ECL) reagents. If fresh tissue is being used, this method is further limited due to the time constraints of needing to isolate individual fibers within a limited timeframe (i.e., 1-2 h). Fortunately, this restraint is mitigated by isolating single-fiber segments from freeze-dried tissue⁸. However, fiber collection from freeze-dried samples is limited by the size and quality of the biopsied tissue.

Fiber type identification using the dot blotting method⁹ has been significantly elaborated upon and expanded in this comprehensive protocol. Previously, it has been demonstrated that as little as ~2-10 mg of wet weight muscle tissue is adequate for freeze-drying and single-fiber MHC isoform protein analysis⁹. Christensen et al.⁹ used 30% of a ~1 mm fiber segment to detect the MHC isoform present by dot blotting, which was confirmed by western blotting. This work showed that by substituting western blotting with dot blotting, the overall costs were reduced by ~40-fold (for 50 fiber segments). Fibers were then "pooled" into type I and type II samples, which allowed for experimental replication⁹. Nevertheless, a limitation was that only two fiber type-specific samples were obtained: type I (MHCI positive) and type II (MHCII positive fibers), with type II samples containing a mixture of MHCIIa and MHCIIx^6,10. Notably, the current protocol demonstrates how pure type IIx fibers can be identified and provides a highly detailed workflow (summarized in Figure 1), including troubleshooting strategies for common protocol issues.

Human muscle samples were obtained from the vastus lateralis from n = 3 (2 males, 1 female), aged 70-74 years old under sterile conditions using local anesthesia (Xylocaine) and a Bergstrom needle modified for manual suction^11,12. Samples were a subset of a previous study approved by the Victoria University Human Research Ethics Committee (HRETH11/221) and conducted in accordance with the Declaration of Helsinki¹³. Participants provided written, informed consent to participate in this study. Full details of all materials required for this protocol are shown in the Table of Materials. In addition, a list of troubleshooting strategies addressing common protocol issues is provided in Table 1.

1. Freeze drying

1. While keeping the biopsied muscle samples frozen on dry ice, weigh and tare an empty frozen tube on the scales.
2. Quickly weigh a minimum of 10 mg of wet weight frozen tissue. Record the weight to one decimal place.
3. Deposit the tissue into the precooled microcentrifuge tube that has had 3-4 holes pierced into the lid and place it into a small beaker with 2-3 pellets of dry ice.
4. Follow the freeze-dryer manufacturer's operating instructions.
5. Ensure that all the valves on the freeze dryer are closed and switch on the freeze dryer.
6. Use the control panel to check that the vacuum set point is programmed for optimal freeze-drying conditions for human tissue, 0.12 millibar (mBar) (Figure 2).
7. Press manual on the freeze dryer and wait until the chamber has cooled to −40 ^oC.
8. Once the chamber has reached that temperature, remove the lid, then the glass chamber, and place the beaker (with muscle sample) onto the metal stage.
9. Replace the glass chamber and lid. Close the vacuum release valve on the lid and wait until the vacuum scale light turns green to ensure the dryer is vacuum sealed.
10. Freeze-dry the muscle samples for 48 h. Once freeze drying is complete, turn off the vacuum pump by pressing the vacuum button and open the vacuum release valve on the lid.
11. Remove the lid of the chamber and collect the freeze-dried sample. Weigh the tissue and record the weight to one decimal place.
12. Calculate the percentage loss of weight; ~75% decrease in weight indicates the tissue is successfully freeze-dried (in this work, mean ± SD, 76 ± 9%, n = 11 muscle samples).
13. Press AUTO to switch off the vacuum pump and freezer. Allow the unit to reach ambient temperature.
14. Clean the cooling coils according to the manufacturer's instructions and drain accumulated liquid from the collector.
    ​NOTE: Fiber collection can commence immediately. When tissue is not being used for fiber isolation, keep the freeze-dried sample at -80 °C in a sealed container with desiccator beads.  CAUTION: Dry ice is hazardous (Class 9); refer to the MSDS for recommended safe handling.

2. Fiber collection

1. Fiber collection preparation
   1. Label a minimum of 50 microfuge tubes (Fiber 1 to 50) and pipette 10 µL of denaturing buffer to each tube.
   2. Prepare the collection area with two pairs of fine tissue dissecting forceps, lint-free tissue, a benchtop lamp, a stereomicroscope (with the black stage up), a vortex, and a benchtop centrifuge.
   3. Place the freeze-dried muscle sample onto a Petri dish lid. Place the lid on the stage of the dissecting microscope.
   4. Watch the video of single-fiber dissection. Practice the complete protocol from fiber collection to single-fiber type identification at least twice before commencing a study.
   5. Prior to fiber collection, calculate the total volume required for a fiber type sample from each biopsy as follows. For example, if the starting volume of 1 fiber = 10 µL and the volume remaining after MyDoBID is (1 fiber × 10 µL) - 1 µL used for dot blotting = 9 µL (sample volume), calculate the total sample volume required by multiplying the sample volume (µL) by the total number of western blots that will be run and double this amount to account for redundancy: (9 µL × 4 western blots) × 2 = 72 µL. Calculate the number of fibers required per typed sample by dividing the final sample volume required (µL) by the sample volume per fiber type-specific sample (e.g., 72 µL ÷ 9 µL = 8 fibers per fiber type-specific sample). For this to be achieved, collect a total of 50 fibers.
      NOTE: The sample volume required is the minimum protein concentration required to run both MyDoBID and western blotting if the amount of protein loaded is equivalent to a ~3 mm fiber (~12 µg of wet weight) for each sample (see Supplementary File 1 for details). However, this volume does not factor in whether repeat gels are required for a given protein.  CAUTION: Forceps are sharp; handle them with care to avoid the risk of skin puncture.
2. Single-fiber isolation
   1. On a small piece of paper 5 cm x 1 cm, use a ruler to draw a 1 cm line. Mark every 1 mm on that line.
   2. Insert this under the Petri dish lid as a guide to estimate the length of the fibers collected.
   3. Place the freeze-dried muscle sample under the stereomicroscope at low magnification (x 7.5). Use one pair of fine tissue dissecting forceps to hold the freeze-dried muscle in place and with the other start to separate small bundles of fibers (as seen in the video).
   4. Isolate one bundle of fibers and continue to tease apart until single fiber segments are isolated from the bundle.
   5. Collect a minimum of 50 fibers that are at least ~1 mm in length.
   6. Move the fiber to an empty space on the Petri dish. Inspect single-fiber segments under higher magnification (x 50). Ensure it is a single fiber by further separating the fiber at the end. If the fiber breaks instead of separating, it is a single fiber.
3. Denaturation of fibers
   1. Using forceps, gently collect the fiber and place it directly into the aliquoted denaturing buffer (do not let the forceps encounter the buffer).
   2. Check the forceps under the microscope to ensure the fiber has been successfully removed. Close the tube and tap the bottom of the tube firmly on the bench three times to ensure that the fiber moves into the buffer.
   3. Wipe the forceps clean using lint-free tissue before moving on to the next fiber. Repeat this process until all fibers are collected.
   4. Vortex the fiber samples and briefly centrifuge for 5 s at 2,500 × g to draw the sample to the bottom of the tube.
      NOTE: The centrifugation duration (5 s) is timed from the start (0 × g) until the speed reaches ~2,500 × g.
   5. Leave the samples at room temperature for 1 h. Store at -80 °C for future use. Freeze and then thaw the samples before use.

3. Dot blotting

1. Membrane preparation and activation
   1. Measure, label, and cut to size one polyvinylidene difluoride (PVDF) membrane (0.2 µm pore size) to fit 50 samples (~10.5 cm x 5.5 cm).
   2. Mark the left border numerically from top to bottom (1 to 10) and the top border alphabetically from left to right (A to E) spaced 1 cm apart.
   3. Prepare four sheets of filter paper with the dimensions of 12.5 cm x 7.5 cm.
   4. Pile two sheets together to form a stack. Presoak the filter paper stack in 1x transfer buffer.
   5. Place the membrane in a container. Pour 95% ethanol over the membrane, with enough volume to entirely immerse the membrane (10-15 mL). Agitate on the rocker for 1 min.
   6. Recollect the ethanol, immerse the membrane in 1x transfer buffer (~15 mL), and rock for a further 2 min.
      NOTE: Both ethanol and transfer buffer can be reused for future dot blotting experiments until sedimentation forms (change after six uses).
   7. Lay the transfer buffer-soaked filter paper stack on a flat movable surface such as a large lid and flatten with the roller. Position the PVDF membrane on the stack using a gel releaser or tweezers.
   8. Place a single sheet of dry filter paper on top of the membrane and move the roller over the filter paper to soak up any excess buffer on the membrane surface. Remove the top filter paper without rubbing the membrane.
      CAUTION: Methanol (Class 3, sub-risk 6.1) and ethanol (Class 3) are hazardous. Refer to the material safety datasheet (MSDS) for safe handling recommendations.
2. Sample absorption to the membrane
   1. Thaw the fiber samples, perform a brief centrifuge (5 s) at room temperature as in step 2.3.4 and mix the sample thoroughly.
   2. Without touching the membrane with the pipette tip, slowly deposit a ~1 µL drop of each sample onto the membrane in the designated area. The dimensions of the designated area per fiber sample are ~1 cm x 1 cm. Aim to spot the sample in the center of this area.
   3. Allow the sample droplets to soak through the membrane for at least 15 min. Ensure no sample buffer remains and is completely absorbed.
   4. Using plastic tweezers, carefully lift the membrane off the damp filter paper stack, place it on a single dry sheet of filter paper, and dehydrate the membrane for at least 5 min.
   5. Once the sample spots turn completely white, reactivate the membrane.
3. Membrane reactivation and block
   1. Reactivate the membrane by repeating steps 3.1.5 and 3.1.6.
   2. Place the membrane in wash buffer and rinse for 5 min with rocking. Discard the wash buffer.
   3. Incubate the membrane in blocking buffer for 30 min while rocking.
   4. Rinse the membrane 3x with wash buffer until the buffer is no longer cloudy.
   5. Leave the membrane in the final wash on the rocker.

4. Immunolabeling

1. Detection of MHCIIa
   1. Dilute the MHCIIa primary antibody at 1 in 200 in 10 mL of Bovine Serum Albumin (BSA) buffer.
   2. Discard the wash buffer from the membrane and then pour the diluted MHCIIa antibody onto the membrane.
   3. Place the container (with the membrane in MHCIIa) on the rocker at room temperature for 2 h or at 4 °C overnight.
   4. Recollect the MHCIIa antibody and store it at 4 °C. Wash the membrane with blocking buffer for 2 min on the rocker.
      NOTE: All primary antibodies can be reused up to five times as long as the buffer remains clear.
   5. Rinse twice more; 5 min each time; three washes in total.
   6. Dilute mouse Immunoglobulin G Horseradish Peroxidase (IgG-HRP) secondary antibody at 1 in 20,000 in 10 mL of blocking buffer and add to the membrane.
   7. Discard the wash buffer and incubate the membrane in mouse IgG-HRP secondary with rocking for 1 h.
   8. Discard the secondary and wash the membrane in wash buffer (2, 5, 5 min).
   9. Leave the membrane soaking in the final wash until ready for imaging.
   10. Turn on the gel imager, wait 15 min for the imager to reach the required operating temperature, and then open the imaging software (see Table of Materials).
   11. In the software window, click New Single Channel Protocol (Figure 3A). For a white light image of the membrane, under Applications | Blots | click Colorimetric (Figure 3B).
   12. Click on the gel area; each option is programmed for specific dimensions to fit the size of various gel types. Select the appropriate option to best fit the dimensions of the membrane (Figure 3C).
   13. Prepare the ECL by combining luminol and peroxidase reagents at a 1:1 ratio. Make a sufficient volume of ECL mixture to cover the entire membrane, typically a total volume of 800-1,000 µL is required.
   14. Place the membrane on a large clear plastic tray and disperse the ECL mixture onto the membrane.
   15. Place the membrane onto the imaging plate.
   16. Click on Position Gel (yellow button) for a live camera view. Click hold and drag the zoom button to maximize the imaging area of the membrane. At this point, straighten the position of the membrane if required. 
   17. Click Run Protocol (green button) and wait for a colorimetric image of the membrane to be produced. Save this image to assist in matching the dots to the corresponding fiber sample.
   18. Without moving the membrane, change the application on the software by clicking the option for a 2 x 2 binning (Chemi High Resolution) (Figure 3D).
   19. Under Image exposure and software, optimize the exposure time, click Faint Bands (Figure 3E) | Signal Accumulation Mode.
   20. Under Setup, type in 1 s for the first image, 30 s for the last image, and 30 total images (Figure 3F).
   21. Click Run Protocol.
   22. Save an image at the following stages: before signal saturation (all visible dots are black), initial signal saturation (some dots begin to saturate), and oversaturated spots (all spots with strong signal detected are saturated).
   23. Save all required images before ending signal accumulation. Take the membrane out of the imager.
   24. Using the gel releaser, place the membrane back into the container with the wash buffer.
2. Detection of MHCI
   1. Pour the wash buffer out and treat the membrane with stripping buffer for 30 min at 37 °C (ensure the membrane is completely immersed).
   2. Recollect the stripping buffer and rinse the membrane in wash buffer for 5 min with rocking.
      NOTE: Stripping buffer can be reused 10x before discarding.
   3. Pour out the wash buffer and this time apply MHCI primary antibody diluted at a starting concentration of 1 in 200 (range: 1 in 200 to 1 in 500). Rock the membrane at room temperature for 1 h.
   4. After incubating in MHCI primary antibody, recollect the antibody and wash the membrane as in steps 4.1.4 and 4.1.5.
   5. Dilute mouse IgM HRP secondary antibody in blocking buffer at 1 in 20,000. Pour out the blocking buffer from the membrane and incubate in the mouse IgM secondary as in step 4.1.7.
   6. Wash and image capture detected MHCI as in steps 4.1.8 to 4.1.24.
      CAUTION: Stripping buffer contains Organo Phosphine 3-5% (Class 8). Refer to the MSDS for safe handling recommendations.
3. Detection of potential MHCIIx using Actin
   1. Strip the membrane once more (steps 4.2.1 and 4.2.2).
   2. Repeat section 4.1, this time applying Actin primary antibody diluted at 1 in 500 in BSA buffer and then rabbit HRP secondary antibody diluted at 1 in 20,000 in blocking buffer.

5. Fiber type identification

1. MHCI, MHCIIa, and Actin signal analysis
   1. Become familiar with the signal intensity panel (Figure 4A). Make a note of steps 5.1.2 to 5.1.4.
   2. A saturated signal intensity qualitatively indicates that the protein detection is strong.
   3. A moderate indicates that the target protein is present at a medium level.
   4. A faint signal indicates little target protein is present.
   5. Use the signal intensity panel to categorize fibers with 'saturated,' 'moderate,' or 'faint' signal intensity (Figure 4A).
   6. Carry out fiber type identification by comparing MHCIIa and MHCI results first (Figure 4B, left and middle blots).
   7. Record fibers with saturated or moderate signal intensity of only one MHC isoform.
   8. If the MHC isoform signal is 'faint,' leave the fiber unmarked (see Figure 4B).
   9. Lastly, where Actin is observed (moderate or saturated signal intensity) in the fibers with no MHCI or IIa detected, record it as a potential type IIx fiber (Figure 4B, right blot).
   10. Record fibers with faint MHC isoform signal but Actin detected (moderate or saturated intensity) as unidentified.
   11. Discard samples with faint or no detection (no fiber collected) for all three target proteins (Figure 4C).
   12. Record any fiber where both MHCI and MHCII signals are detected with a 'saturated' or moderate signal. These samples are not for use in fiber type-specific preparation.
2. Preparation of fiber type-specific samples
   1. Prepare a separate type I and type II sample for each biopsy. For a type II sample, combine the minimum required number of fibers (calculated in step 2.1.5) in which MHCIIa is detected at saturated or moderate levels.
   2. In a separate tube labeled as type I, repeat this process for fibers in which MHCI is detected (Figure 4D).
   3. Use fibers with moderate signal intensity if there are not enough fibers with saturated signals.
   4. Combine any potential IIx fibers.
   5. Immediately use fiber type-specific samples for western blotting or store them at -80 °C for future use.

6. Western blot fiber type confirmation

1. Utilize 10 µL of each fiber type-specific sample, (the minimum sample volume required, see step 2.1.5).
2. Separate samples using the specified precast gel via SDS-PAGE according to the manufacturer's protocol.
3. Use a gel imager to detect the protein in the gel according to the manufacturer's protocol.
4. Place the gel into cold 1x transfer buffer for 10 min.
5. Wet transfer the proteins onto a 0.45 µm nitrocellulose membrane (9.5 cm x 13.5 cm) according to the manufacturer's protocol.
6. After transfer, briefly rinse the membrane with ultrapure H₂O in a container. Pour out the ultrapure water.
7. Add 10 mL of antibody signal enhancer solution to the membrane and rock at room temperature for 10 min.
8. Recollect the antibody signal enhancer solution and wash 5x (5 s per wash) with ultrapure H₂O.
   NOTE: Antibody signal enhancer solution can be used 5x before discarding.
9. Pour out the last wash, add blocking solution, and rock at room temperature for 1 h.
10. Use a clean scalpel blade or scissors to cut horizontally across the membrane at a molecular weight that ensures that proteins that are 180 kDa and above are on the top section of the membrane.
11. Use this top section for further fiber type confirmation.
12. Use the portion below 180 kDa to detect different target proteins.
13. On the top section of the membrane, follow section 4.1 with MHCIIx primary and mouse Immunoglobulin M (IgM) HRP secondary antibody.
14. Strip the membrane (as described in sections 4.2.1 and 4.2.2) before repeating the process with MHCIIa IgG primary and mouse IgG HRP secondary antibody.
15. Strip once more and finally apply MHCI IgM primary and mouse IgM HRP secondary antibody.
    NOTE: This step is qualitative and so any protein loss due to membrane stripping is not a concern.
16. Compare the signal detected across the different MHC isoforms (as seen in Figure 5B). When the signal intensity is detected at moderate to saturated levels in the expected sample (MHCI - type I, MHCIIa - type II and MHCIIx - type IIx) the fiber type-specific samples will be recorded as reliable for use in future studies.
17. Record the sample as unreliable when more than one MHC isoform is equally detected in a sample.
    CAUTION: Antibody enhancer solution contains sodium hydroxide 50%. Refer to MSDS for safe handling recommendations.

Identification of individual MHCI, MHCIIa, and MHCIIx muscle fibers using dot blotting
A feature of MyDoBID is the categorization of the varying MHC and Actin signal intensity strength in a given fiber (Figure 4A). Fiber type was identified by the presence or absence of MHCI and IIa isoforms (Figure 4B). Six fibers showed no MHC or actin detection, indicating that there was no fiber collected. The results of this specific dot blot were the identification of 22 type II fibers, 7 type I fibers, and 3 potential type IIx fibers; 2 unidentified samples and 6 samples were classified as 'No Fiber' collected (Figure 4B, C). Fibers that did not have any detectable MHCI or MHCIIa signal yet were positive for Actin were identified as potential type IIx fibers by a process of elimination. Using the signal intensity panel to classify MHCI and IIa signal strength, 'moderate' or 'saturated' fibers were combined to produce type I or II samples, respectively. Fibers that were unidentified or had faint or no Actin detected were not included in subsequent analysis and were discarded (Figure 4D).

Fiber typing confirmation using western blotting
The fiber type-specific samples were validated using western blotting and MHC-specific antibodies (Figure 5). Type I and type II fiber samples from one individual biopsy were run along with a type IIx sample, which was a sample of potential type IIx fibers from two individuals due to the low numbers of type IIx fibers present in each biopsy. Western blot data confirmed that the fiber type identification was successful.

Figure 1: Workflow summary outlining sample preparation, fiber typing, and western blot confirmation of fiber specificity. (A) Human tissue is freeze-dried for 48 h. (B,C) Single fiber segments are isolated under a dissecting microscope. (D) Fibers are denatured and (E) after 1 h, stored at -80 °C. (F) Primary antibody incubation order. (G) Fibers were dot blotted and fiber type identified using MHCIIa (type II), MHCI (type I), and actin (IIx?, potential type IIx fiber). (H) Fibers were then pooled. (I) Fiber type-specific samples alongside a calibration curve are analyzed via SDS PAGE and western blotting. (J) Validation of fiber type identification using western blotting. Please click here to view a larger version of this figure.

Figure 2: Display screen and control panel of the freeze-drying system. Please click here to view a larger version of this figure.

Figure 3: Imager settings for white light capture of the membrane followed by chemiluminescent detection of target proteins. To image the membrane under white light, (A) select New Single Channel followed by (B) Blots | colorimetric application. (C) Based on the size of the gel used, select the appropriate imaging area, and then take a white light image by pressing Run Protocol. (D) Without moving the membrane, repeat step B but this time, choosing Blots | Chemi Hi Resolution. Before pressing run, choose (E) Image Exposure to optimize for Faint Bands and (F) set up the Signal Accumulation Mode and in the first instance, set the exposure time for every second for a total of 30 s with Highlight saturated pixels ticked. Press Run Protocol and allow the run to complete before selecting the best images for saving. Please click here to view a larger version of this figure.

Figure 4: Fiber type identification. (A) Fiber types were identified relative to all other fibers on the membrane, categorized by the signal intensity panel for each MHC isoform detected (e.g., MHCI signal: Saturated, Moderate, and Faint). (B) Using the panel in (A), the MHCIIa antibody identified type II fibers (blue, left blot), and after stripping the membrane, the MHCI antibody identified type I fibers (red, middle blot). Actin antibody was used to identify potential IIx fibers if a positive Actin signal was detected in fibers where no previous MHCI or IIa signal was detected (green, right blot). Fibers that did not follow these guidelines are highlighted in purple (unidentified). Lastly, samples negative for the Actin signal indicated that no fiber had been collected ('No Fiber'). (C) Table showing fiber labeling corresponding to the sample position on dot blots shown in (B). (D) Outline of fiber selection for preparing fiber type-specific samples. The aim was to collect 10 saturated fibers for each fiber type. Here, for type II fibers, eight saturated fibers were identified and pooled. For type I fibers, less than 10 saturated fibers were present, so fibers with moderate signal strength were also selected. For type IIx fibers, two fibers were identified and pooled. If less than 10 fibers (saturated and moderate) are identified, further fiber collection may be necessary.  NOTE: The Actin signal shown in (B) has not reached saturation. Please click here to view a larger version of this figure.

Figure 5: Western blot confirmation of fiber type identification. Fiber type identification was carried out as per Figure 4 to generate type I (~10 fibers) and type II (~10 fibers) samples. As there were no IIx fibers identified from the same biopsy, the IIx sample shown here was produced from more than one muscle sample (n = 2 individuals). A small aliquot of each fiber-specific sample is analyzed by western blotting to confirm fiber type identification. Molecular weight markers (kDa) are indicated on the left, captured under white light without moving the membrane. Target proteins are detected in the sequence of MHCIIx, MHCIIa, and MHCI, with the myosin from the UV-activated gel visually indicating the amount of protein loaded per sample. Please click here to view a larger version of this figure.

Table 1: Troubleshooting strategies for common protocol issues. Please click here to download this Table.

Supplementary File 1: Calculation of freeze-dried fiber mass using a calibration curve. Please click here to download this File.

Fiber collection
Based on several years of experience, most researchers can master this technique; however, practice leads to faster and more efficient fiber collection for downstream analyses. To be able to isolate 30 single fiber segments of a quality for pooling, it is recommended that 50 fiber segments are collected per sample. Studying the fiber collection video carefully and after performing two practice sessions (~50 fibers per session) is recommended to achieve a reasonable standard. All fibers collected undergo MyDoBID to identify the fiber type, which will reveal how effectively the technique is being performed. This would be seen as a low number of 'No fiber' or 'faint' MHC signal detected in fiber samples (see protocol section 5.1).

It is known that muscle can contain hybrid fibers, where both MHCI and MHCIIa are present, or MHCIIa and IIx^2,14. A limitation of the MyDoBID is that the presence of a hybrid fiber cannot be ascertained because a sample positive for both MHCI and MHCIIa antibodies could be either a hybrid fiber or that two fibers were collected. Further, MyDoBID cannot detect MHCIIa separately from MHCIIa/IIx hybrids as the MHCIIa primary antibody would detect the presence of MHCIIa in both IIa and IIa/IIx fibers. For these reasons, identifying and preparing samples of hybrid fibers was not performed, and samples that have both MHCI and MHCIIa signals that fell outside the ranges described in the signal intensity panel were discarded (Figure 4A). A further limitation is that the proportion of muscle fiber types in a sample cannot be determined using MyDoBID, and applying conventional immunohistochemical analyses is necessary to determine fiber type distribution².

Dot blotting
The signal intensity panel and fiber type ID rely on the entire sample being represented in the small volume applied to the dot blotting membrane. To accomplish this, the sample is thoroughly mixed by agitation before loading onto the membrane. All fibers with only one MHC isoform move to the next step of pooling. At this stage, it is important that any fiber with more than one MHC must not proceed to this next step of combining samples. If a sample is contaminated, then the whole pooled sample needs to be discarded and the researcher must go back to Fiber collection and repeat the fiber isolation and dot blotting steps.

Immunolabeling
Previously it was reported that 5 min is an adequate time to block non-specific sites on the membrane for the detection of MHCI and MHCIIa⁹. Advancing the technique to MyDoBID requires an Actin antibody to be used. It was found that the 5 min block time needed to be increased to 30 min, as reported⁶, to obtain a membrane with minimal background, as 5 min blocking resulted in a darker membrane background. Blocking time is a consideration in any immunodetection technique, such as western blotting or immunohistochemistry, and should be modified as appropriate.

Stripping the membrane
Stripping cannot be used for quantitative analyses¹⁵ because it also removes proteins of interest but can be used in MyDoBID since it is a qualitative assay. The purpose of stripping is to remove the bound antibodies from the surface of the membrane, allowing a new antibody to be used with minimal interference from the prior antibody. In MyDoBID, there is the option of applying the sample across two membranes, where the different MHC isoforms are detected on separate membranes. If this approach is preferred, avoid handling the sample tube more than once by loading the spots at the same time. This will ensure that the drying time between each sample spot is similar (~5 s difference) and subsequent steps performed simultaneously in separate containers. It must be noted that this option would increase the time, sample, and consumable consumption.

Antibodies are applied to first detect MHCIIa fibers, followed by MHCI, because the MHCI antibody always gives a very bright signal, which would be harder to strip than the MHCIIa. Actin is homogeneously expressed in skeletal muscle fibers and is used to confirm that a portion of the fiber was successfully applied to the membrane as described above.

Imaging, fiber type identification, and preparation of fiber type-specific samples
A new tool has been developed in this protocol to assist in fiber type identification and sample selection to create fiber type-specific samples (Figure 4A). The example shown in the signal intensity panel is used to define MHCI signal intensity as: (i) saturated, (ii) moderate, or (iii) faint. For pooling fibers into fiber type-specific samples, fibers showing saturated signal intensity are selected first and if needed, fibers with moderate signal intensity are added. When preparing type I and II samples, there are typically enough 'saturated' and 'moderate' fibers so that fibers categorized as 'faint' or unidentified can be discarded.

Novelty of using Actin instead of MHCIIx antibody to identify pure type IIx fibers
Previously, to identify pure IIx fibers, the MHCIIx signal was subjectively compared to the MHCI and MHCIIa signal on a dot blot, and by a process of elimination, the fibers with no MHCI or IIa signal, but with MHCIIx signal, were identified as pure IIx. However, issues can arise when using two mouse IgM primary antibodies (MHCIIx and MHCI) on the same membrane. Despite stripping the membrane between primary antibodies, residual antibody signal may be detected. Utilizing rabbit anti-Actin primary antibody prevents this issue from occurring. Here we used Actin to confirm that a fiber was collected and in the absence of both MHCI and MHCIIa, indicates that a IIx fiber was present. Potential IIx fibers are then confirmed by western blotting using the MHCIIx-specific antibody (see Figure 5 in this paper and Figure 2 in⁹). The 6H1 antibody has been shown¹⁶ to detect the electrophoretically separated band corresponding to MHCIIx in rat skeletal muscle and labels type IIx fibers in cross-sections of mouse, rat, and human muscles².

Western blot fiber type confirmation
It is best practice to run all fiber type-specific samples from the same biopsy on the same gel. With MHCI antibody requiring the same secondary antibody as MHCIIx, type I and IIx samples should be separated by at least one lane on a given gel, as shown in Figure 5. As the protein content will vary across fiber type-specific samples due to variable fiber size, the first gel should be used to determine the volume of sample that needs to be loaded in subsequent gel runs to attain similar amounts of protein across all samples. To do this, the total protein in a given sample is determined using UV-activated gel imaging technology to visualize all protein bands that have been separated in the gel by SDS-PAGE prior to transfer. The total protein in each sample is determined from the gel image and used to correct unequal loading in the next western blot run, provided a calibration curve of several known masses of skeletal muscle tissue is run alongside the fiber type-specific samples^3,4,6,17. There is also no need to use a 'housekeeping' protein such as Actin to determine the total protein, which would also require its own calibration curve¹⁵. This technology is a timesaving and accurate method to determine the total protein and can be performed with very little sample (i.e., ~1/5^th of a fiber⁹) without the need for additional reagents or protein standards while maximizing all sections of the membrane for detecting proteins of interest.

When it comes to immunolabeling during the western blot step, the MHCIIx antibody is applied first due to the relatively low abundance of MHCIIx compared to the other MHC isoforms. Once the membrane is ready for MHCI antibody detection, it would have undergone two steps of stripping (i.e., post MHCIIx and MHCIIa detection). Consequently, the detection of MHCI should primarily be detected in a type I sample, with minimal residual signal detected from previous MHC antibodies (Figure 5). Western blotting is used to confirm the purity of each fiber type-specific sample using all three MHC antibodies on the same membrane portion. However, the need for stripping would be eliminated if either MHCI or MHCIIx antibodies were raised in a different host species such as rabbit or goat.

Applications
The methodology described here can be utilized for fiber typing single fiber segments from fresh or freeze-dried skeletal muscle to examine fiber-type specific protein expression in human skeletal muscle. In addition, using fresh muscle samples, either intact or mechanically skinned fibers can be used⁶. Using a skinned fiber allows further examination of protein expression in subcellular compartments such as those in the cytosol and membrane compartments. This application has been previously used to measure the amount of glycogen that is bound vs. unbound in mechanically skinned fiber segments from healthy and diabetic individuals⁶. Importantly, MyDoBID is beneficial for experiments that utilize denatured samples only and would not be appropriate for studies that require proteins in their native state. While it is possible that other analyses such as enzymatic assays or in-solution proteomic approaches could be performed under native conditions, further optimization would be required so that a portion of the fiber could be removed for MHC isoform identification using MyDoBID.

In summary, type I, type II, and type IIx can be successfully identified by the application of a straightforward technique with readily available consumables commonly used for western blotting. We include extensive practical information, where upon successful completion will produce fiber type-specific samples that are of the best possible quality, integrity, and reliability for future studies. This technique is the practical and preferred approach to performing fiber-specific skeletal muscle biochemical analysis.