Single Fiber Isolation Assay for the Assessment of Oxidative Myofiber Behavior

Summary

This protocol describes the isolation of intact single muscle fibers from the oxidative soleus and the establishment of highly purified myoblast cultures derived from these fibers. The optimized procedure reliably yields approximately 300–500 intact myofibers per mouse, enabling downstream molecular and cellular analyses.

Abstract

Skeletal muscle is composed of multinucleated myofibers whose integrity and function are essential for movement and overall health. In neuromuscular disorders, muscle groups can be affected differentially across disease types and stages, making it important to isolate viable single fibers from representative muscles for mechanistic studies. The objective of this protocol is to provide a reliable method for isolating a high yield of intact single myofibers from the murine soleus (SOL) muscle, which is challenging to dissociate due to fiber length and fragility. By optimizing collagenase concentration and digestion time, the protocol minimizes fiber loss and preserves viability during dissociation. The optimized workflow markedly reduces residual tissue attachment to isolated fibers, thereby limiting carryover of non-fiber cells and minimizing cross-cell contamination. This technique is suitable for single-fiber analyses as well as satellite cell isolation and subsequent expansion under conditions with minimal contamination. Using this optimized workflow, we routinely obtain ~300–500 intact SOL-derived single myofibers that can be immunolabelled with high efficiency and also yield a high-quality satellite cell population with minimal fibroblasts contamination. Using comparative analyses with Extensor Digitorum Longus (EDL) muscle we underscore the value of a tailored, muscle-specific approach to evaluating therapies in neuromuscular disease research, enabling candidate pharmaceutical interventions to be tested within small, tunable experimental windows.

Introduction

Skeletal muscle is the most abundant tissue in the human body, accounting for approximately 40–50% of total body mass¹. Skeletal muscle serves as a reservoir of energy in the form of glycogen. During cold exposure, it provides heat through thermogenesis and importantly provides locomotion. Skeletal muscle is composed of multinucleated, post-mitotic myofibers. To sustain muscle maturation and repair, new myonuclei are generated by our skeletal muscle's resident stem cell, the satellite cell (SC). SCs are found on the periphery of postnatal skeletal muscle fibers^2,3,4. SCs are identified by the transcription factor Pax7, which is expressed in both quiescent and activated states^3,5. Upon injury or hypertrophic stimulation, SCs activate and upregulate MyoD, enter the myogenic program, proliferate, and ultimately fuse with existing or newly forming myofibers to restore muscle function^6,7,8. SCs are heterogeneous in their stemness ability, embryological origin (somitic vs non-somitic), stage of postnatal growth (young vs old), and muscle composition (fast vs slow fibers)^9,10,11.

Depending on the region of the body, skeletal muscle is tailored to meet the energetic and functional demands of that region. This heterogeneity in muscle fibers is possible due to their contractility and oxidative capacity. Indeed, muscle fibers are composed of slow-twitch oxidative fibers (termed type I and IIa) and fast contracting glycolytic fibers (termed IIx, in rodents IIb)^12,13. This beautiful, functional mosaic of fiber types enables us to move freely in our everyday lives. For example, postural muscles such as the soleus (SOL) and erector spinae are in a state of chronic, low-level activation, and thus contain a high proportion of slow-oxidative type-I and type-IIa muscle fibers. In contrast, short but fast-acting muscles such as the extensor digitorum longus (EDL) are mainly composed of glycolytic type-IIx and type-IIb fibers. The fiber composition, however, is not fixed; rather, muscle fibers are dynamic structures capable of shifting from fast-to-slow and vice versa depending on the functional requirements placed upon them. The difference in fiber type contractility is possible due to the content of myosin protein. This is the main protein within skeletal muscle, composed of isoforms of myosin light and heavy chains^12,14.

Inside a sarcomere (muscle’s smallest functional unit that generates force), myosin heads and actin interact to form a structure known as a crossbridge. Through hydrolysis of ATP, energy is released by the cycling of the myosin and actin interactions^15,16. The Myosin heavy chain (MyHC) is the most appropriate marker for fiber type identification^12,13. Depending on the isoform of MyHC expressed, the rate of muscle contractility will vary. As mentioned, type-I fibers are slow oxidative fibers, unlike type-II fibers. Type-I dominant muscle groups contain a higher abundance of oxidative enzymes, are richer in mitochondria, and have a dense capillary network to perfuse the muscle^17,18 (think marathon runner). Type-I fibers are encoded by the MYH7 gene^13,19. Type-II fibers are fast to fatigue, contain a high proportion of glycolytic enzymes, and have a greater maximum velocity of shortening, making them ideal for short bursts of power or movement (think power lifter). Type-II fibers for type-IIa, IIx, and IIb (in rodents) are encoded by the MYH2, MYH1, and MYH4 genes, respectively^20,21. Unsurprisingly, any pathophysiological insult that affects muscle integrity will alter fiber type composition and lead to detrimental effects on muscle function, potentially lowering quality of life. In fact, many muscular dystrophies either start in the satellite cell (satellite cell-opathies) or in the muscle fiber (motor neuron- or muscle tissue-derived), and can cause a shift in fiber type composition^22,23,24,25. For example, Spinal cord injury, bedrest studies, and spaceflight (microgravity) show that long-term inactivity can trigger postural muscles to shift from a slow to a fast fiber phenotype^{26,27,28,29,30}. Conversely, in Duchenne muscular dystrophy, both in human and mouse models (mdx), fast glycolytic fibers are affected to a greater extent and with greater severity than their slower counterparts^31,32,33,34. Similarly, in facioscapulohumeral muscular dystrophy (FSHD), a fast-to-slow twitch transition is observed with a greater proportion of type-I fibers along with lower force production in type-II fibers^24,35,36.

It remains unclear why certain muscle fiber types are selectively affected or spared in different diseases. The fact that these patterns are non-random underscores the importance of dissecting the underlying mechanisms, for which single-fiber isolation has emerged as a powerful and widely used technique. Indeed, since first introduced in the mid-80s by Bischoff³⁷, this indispensable tool has been continually refined and adapted, enabling increasingly precise investigation of muscle biology^38,39,40. Importantly, single-fiber isolation maintains SCs as close as possible to their physiological niche, enabling adequate scrutiny of their molecular activity. Although muscle groups such as those from the flexor digitorum brevis (FDB) and EDL can be extracted with relative ease^41,42,43, the SOL muscle remains one of the most difficult muscles to obtain a rich population of intact fibers from. This is due to its rigidity, long fiber length, and susceptibility to damage. To address this, a technique has been optimised in the present study that robustly yields a high-quality, viable population of fibers from the notoriously difficult-to-isolate SOL muscle. When combined with EDL fiber isolation, this approach enables culture of fibers and derivative cells (satellite cells, myoblasts, and myotubes) from distinct muscle groups. Critically, this platform allows systematic investigation of the molecular factors that may differentially contribute to diverse muscle pathologies and provides a powerful means to test pharmaceutical interventions in a way that can reveal muscle-type-specific responses and ultimately support the development of effective treatments for skeletal muscle diseases.

Protocol

All animals were housed in the animal facility of Tokyo Metropolitan University (Permit Numbers: A4-4, A5-9, A6-7, A7-018). All animals were maintained under specific pathogen free (SPF) conditions. Animal handling and experimental procedures employed in this study strictly adhered to the guidelines of the Safety and Ethics Committee of Tokyo Metropolitan University.

1. Reagent Preparation

1. Wear gloves throughout. In a 25 mL centrifuge tube, prepare 0.5% of Collagenase type I solution in high glucose Dulbecco’s Modified Eagle Medium (DMEM) + GlutaMAX with 1% penicillin-streptomycin-amphotericin B suspension (AB). After collagenase is well dissolved (lightly tap the tube if needed), sterilize the solution by filtering through a 0.22 µm low-protein-binding filter.
   NOTE: 2.5 mL of collagenase solution is enough for two soleus (one mouse).
2. Prepare 10 mL of 5% bovine serum albumin (BSA) in phosphate-buffered saline (PBS). Heat-inactivate at 60 °C for 30 min, then filter the solution through a 0.22 µm low-protein-binding filter.
   NOTE: The 5% BSA in PBS solution can be stored at 4 °C for up to 10 days.

2. Dissection of the soleus muscles

1. Layout equipment for muscle dissection (Figure 1A).
2. Anesthetize C57BL/6 mice by intraperitoneal injection of a triple anesthetic mixture (0.5 mL/g body weight) containing 1 mg/mL medetomidine hydrochloride, 5 mg/mL midazolam, and 5 mg/mL butorphanol tartrate, then euthanize the mice by cervical dislocation.
3. Make sequential skin incisions on the hindlimb and peel away the skin. Remove the surface fascia.
4. Separate the plantaris (PLA) tendon from the gastrocnemius (GAS) and soleus (SOL) tendon bundles. Gently remove the PLA by pulling from its tendon (Figure 1B).
5. Cut the GAS and SOL tendon bundles and expose the muscles. Insert a pin into the gap between the proximal tendon of the GAS and SOL (Figure 1C).
6. Carefully slide the needle toward the distal end to separate SOL and GAS muscle (Figure 1D). Place a second needle adjacent to the ankle joint to stabilize the hindlimb and prevent movement during dissection.
7. Cut the tendon bundles from the GAS side (Figure 1E).
8. Rest the SOL on the GAS. Carefully adjust the position of the SOL to expose the proximal tendon and cut it with a pair of micro scissors (Figure 1F).
9. Position the SOL on the dissection table and remove the remaining tendon (Figure 1G).
   NOTE: This step is optional; the remaining tendon does not affect the outcome.

3. Digestion of the soleus muscles

1. Transfer the intact soleus into a 25 mL centrifuge tube containing the prepared 0.5% collagenase solution. Incubate at 37 °C for 130 min.
   NOTE: Every 15–20 min, one can manually agitate the sample to slightly separate fibers and support collagenase activity.

4. Isolation of single fibers under a stereomicroscope

1. Cut two Pasteur pipettes to create one with a wide bore and one with a narrow opening (Figure 2A). Under flame, sterilize and smooth the holes to avoid muscle damage during trituration.
2. To prevent fiber adhesion during isolation, coat Pasteur pipettes and three 50 mm Petri dishes with 5% BSA/PBS.
   NOTE: A stereomicroscope (Figure 2B) was used throughout the isolation steps.
3. After muscle digestion, discard the excess collagenase solution and transfer the soleus into the first dish containing DMEM with 1% AB (Figure 2C).
4. Using the wide-bore Pasteur pipette, gently rinse up and down to dissociate fibers (Figure 2D).
5. With the narrow-bore Pasteur pipette, transfer intact single fibers along the muscle fiber length to the second dish. Take the opportunity here to remove debris and/or shrunken fibers (Figure 2E,F).
6. Transfer high-quality (full-length/intact) single fibers to a third dish to ensure purity with the narrow-bore Pasteur pipette (Figure 2G,H).
7. Collect the fibers into a BSA-coated 25 mL centrifuge tube. Gently stir the suspension, then leave it for 5 min at room temperature to allow the fibers to precipitate to the bottom of the tube.
8. Using the narrow-bore Pasteur pipette, carefully discard the supernatant, removing any remaining debris. Leave approximately 500 µL of the DMEM with 1% AB solution. This precipitate will contain a highly purified population of single muscle fibers (Figure 2G,H).
   NOTE: This technique can yield > 300 single fibers, which can then be used for protein and mRNA extraction and subsequent analysis.

5. Establishment of highly purified myoblast cultures from soleus fibers

1. Dilute Matrigel Matrix to 0.1 mg/mL and use it to coat a 100 mm culture dish. Incubate the dish at 37 °C for 30–60 min until the coating polymerizes evenly.
2. Add an equal volume of Accutase to the muscle fibers suspension and incubate at room temperature for 10 min. Subsequently, add 10 mL of growth medium (composed of glucose-free DMEM containing 30% fetal bovine serum, 1% GlutaMAX, 1% chicken-embryo extract, CEE, 1% AB, and 0.01 µg/mL basic fibroblast growth factor, bFGF)⁴⁴.
   NOTE: An Equal amount of Accutase in this case is the 500 µL described in Section 4.8. Accutase facilitates the gentle dissociation of muscle fibers and helps release satellite cells, supporting the subsequent expansion of the myoblast culture.
3. Transfer the 10 mL of Accutase-treated muscle fiber suspension into a Matrigel-coated dish. Approximately 4 single fibers/cm² are added to a 100 mm culture dish. Ensure the fibers are evenly spread throughout the culture dish.
4. After 3 days in culture medium, myoblast colonies can be observed. Replace the growth medium daily until myoblasts reach the required population. Normally, by day 6, myoblast cultures become confluent enough for the experimental phase (Figure 3A).
5. Detach myoblasts from their original culture dish using Trypsin and incubate the sample at 37 °C for 2 min. Using a hemocytometer, count the cells and seed according to the well size and the cell confluency required for myogenic progression.
   NOTE: For a 100 mm dish, add 1 mL of trypsin.

Results

Our technique demonstrates that isolated fibers of the soleus (SOL) and extensor digitorum longus (EDL) muscles can be immunolabelled efficiently against Pax7 (Figure 3B). These isolated fibers can be kept in a floating culture medium (composed of DMEM + GlutaMAX with 10% horse serum, 1% AB, 0.5% CEE) for 3 days, followed by immunolabelling (Figure 3B). Keeping floating fibers for longer than 4 days is not recommended. Under the described floating fiber conditions, myofibers tend to aggregate after this time, compromising subsequent immunolabelling analysis.

The presented technique can further be used for immunoblotting across the myogenic program (Figure 3C). Indeed, SOL- and EDL-derived myoblasts were plated into 24-well plates at approximately 70% confluence. The cells were either maintained in growth medium for 1 day (myoblasts) or induced to differentiate for two or four days (early-forming and differentiated myotubes, respectively). Five µg of protein was loaded for immunoblotting analysis.

MyHC I expression was almost specifically expressed in SOL-derived muscle fibers, with minimal expression in myotubes and negligible in myoblasts (Figure 3C). MyHC II was expressed in both SOL and EDL muscle fibers. This expression reflects the SOL, composed mainly of type-I and IIa, while the EDL is composed primarily of IIx and IIb fibers. Myoblasts did not express MyHC II, but its expression increased progressively during differentiation. The oxidative-related proteins PGC-1α and COXIV demonstrated the expected higher expression in SOL-derived fibers. Also, a higher expression of GLUT4 was detected in isolated SOL fibers compared to EDL fibers. An issue that skeletal muscle biologists may face is that widely used housekeeping proteins and loading controls, such as α-tubulin, β-actin, GAPDH and total protein stains including Ponceau S do not remain stable across myoblasts, myotubes, and isolated muscle fibers, with each showing distinct variability. The signal intensity of α-tubulin and β-actin was highest in myotubes, while Ponceau S staining was relatively weak in myoblasts. In contrast, GAPDH showed stronger expression in EDL muscle fibers. Nevertheless, depending on the experimental purpose, one of these housekeeping proteins may still be applicable, alternatively normalization factors such as heat shock protein 70 (HSP70) can be explored.

Figure 1. Dissection of soleus muscle. (A) Equipment required for dissection. 1: micro scissors, 2: fine forceps, 3: dissecting scissors, and 4: dissection pins. (B) PLA tendon was cut from GAS and SOL tendon bundles (yellow arrow). The distal PLA (white arrow) tendon was lifted with forceps. (C) A dissection pin was inserted into the space between the proximal tendon of the SOL (red arrow) and GAS (green arrow). (D) SOL was separated from GAS using a dissection pin. (E) GAS and SOL tendon bundles were then cut at the GAS side. (F) SOL proximal tendon was then cut with micro scissors. (G) Additional distal tendon bundles were removed. Please click here to view a larger version of this figure.

Figure 2. Isolation of soleus fibers. (A) Equipment required for isolation. 1: narrow-bore and 2: wide-bore Pasteur pipettes. (B) Stereomicroscope used for isolation. (C) Collagenase-digested soleus was transferred to DMEM with 1% AB. (D) Soleus fibers dissociated from the muscle after the first 10 rinse sessions. (E) Single fibers transferred to second dish. (F) Image showing clean fibers removed of debris and shrunken fibers. (G) Debris-free single fibers transferred to the third dish. (H) Intact single fibers clearly visible. Scale bar = 10 mm. Please click here to view a larger version of this figure.

Figure 3. Expansion of cell culture and analysis of EDL and SOL-derived samples. (A) Following 6 days after isolation, the proliferation medium cultured samples show a pure myoblast culture obtained from isolated soleus fibers. Scale bar = 100 µm. (B) SOL and EDL-derived single fibers, immediately or 3 days after isolation, show satellite cells immunolabelled for Pax7 (green). The absence of MyoD (red) confirms that satellite cells have not yet committed to myogenic activation. Fibers were counterstained with DAPI (blue). The cells were subsequently observed through a fluorescence microscope. Scale bar = 20 µm. (C) 5 µg of protein was used for immunoblotting of myoblasts, myotubes, and myofiber derived samples. Immunoblotting panels show expression of MyHC I, MyHC II, PGC-1α, COX IV, GLUT4, α-tubulin, β-actin and GAPDH were used as housekeeping/normalising targets. Ponceau S stain was used as a total protein loading control. Please click here to view a larger version of this figure.

Discussion

The first key to isolating a large number of muscle fibers from the soleus (SOL) is to maintain the structural integrity of the muscle during dissection. If the SOL’s epimysium is torn or if the muscle is pulled forcefully, especially along its longitudinal axis, during dissection, the isolation yield will be severely compromised. These dissection errors are usually easy to identify afterward. Indeed, after collagenase digestion, the muscle becomes uniformly shrunken and forms a white, compact mass. Under such conditions, only a very small number of viable fibers can be dissociated.

The second decisive factor for successful soleus isolation is the efficiency of the type I collagenase digestion. When collagenase is working optimally, approximately 100–200 soleus fibers can be obtained during the first 10 rinse sessions. In contrast, when digestion is suboptimal, even after the first 20 rinses, less than 50 fibers may be released. To obtain a higher fiber yield, more vigorous rinsing and additional rinse cycles are required. However, because SOL fibers are exceptionally fragile, this often results in fiber shrinkage during rinsing.

For establishing a pure SOL-derived primary myoblast culture, collagenase digestion again plays a crucial role. When digestion is efficient, fewer fibers remain attached to residual tissue and transferring the fibers into culture medium results in minimal fibroblast contamination. In contrast, when the SOL is poorly digested, dissociated fibers will remain attached to residual tissue, contaminating the myoblast culture with fibroblasts. Researchers should look for the typical signs of fibroblast contamination in the seeded cell samples; these include much larger cell size and extensive cytoskeletal protrusions, compared to the much smaller, spindle-shaped satellite cells in adhered Matrigel-coated dishes. Should contamination occur, researchers may subject cells to pre-plating steps as described elsewhere⁴⁵. Briefly, SCs are cultured for 3 days post-isolation, then trypsinized and briefly pre-plated on collagen-coated dishes. During 15–30 min of incubation, fibroblasts adhere while SCs remain suspended and are transferred to fresh Matrigel-coated plates.

As mentioned above and consistent with our experience, SOL fibers are longer and more fragile than fibers from the extensor digitorum longus (EDL) and flexor digitorum brevis (FDB). This makes transferring fibers using Pasteur pipettes challenging. It is essential to aspirate fibers along their longitudinal axis rather than drawing them from the middle; otherwise, the fibers fold and shrink under the stress. Aside from the decisive factors that directly determine whether fiber isolation succeeds, a few additional technical steps are essential for improving soleus fiber yield. One of these is to coat all Pasteur pipettes, dishes, and collection tubes with 5% BSA. This prevents freshly dissociated muscle fibers from adhering to surfaces, thereby preventing fiber folding and subsequent shrinkage. It is recommended to prepare new heat-sterilised, BSA-coated Pasteur pipettes each time muscle samples are isolated. Alternatively, researchers could prepare a set of Pasteur pipettes for long-term use. If so, heat-sterilise each time and freshly coat with BSA before use.

The conventional protocol for single muscle fiber isolation was primarily optimized for glycolytic muscles such as FDB and EDL³⁷. However, oxidative muscles, such as the SOL, are structurally distinct. Transcriptomic analyses have shown that extracellular matrix (ECM)-related gene expression differs between the SOL and EDL⁴⁶, including genes such as dystrophin, a cytoskeletal linker protein of skeletal muscle⁴⁴. Consequently, the standard condition for EDL muscle digestion with 0.2% (2 mg/mL) collagenase for 60–90 min is not optimal for SOL muscles, resulting in compact fiber bundles or shrunken fibers after digestion. In this study, we optimized several key steps to adapt the protocol for oxidative SOL muscles. By adjusting the type I collagenase concentration to 0.5% (5 mg/mL), extending the incubation period to 130 min, and minimising mechanical stress during rinsing, 300–500 intact fibers from a single mouse can be consistently obtained. Compared to EDL, the fiber number and yield of SOL samples remain less (the EDL can yield > 500 fibers); nevertheless, the fiber number and, importantly, the high quality of our SOL-derived fibers are robust and suitable for downstream applications such as immunolabelling and immunoblotting. Although our immunoblotting results consisted of pooled single fiber samples, the technique presented in this report is able to reliably extract intact individual fibers, making them suitable for downstream applications such as immunoblotting, proteomics, and RNA-sequencing⁴⁷ at single-fiber resolution. Researchers in the field can use this technique to study metabolic and contractile differences between oxidative and glycolytic muscle fibers.

Overall, this protocol provides a reproducible and accessible method for isolating intact oxidative fibers from the soleus muscle, expanding the toolkit available for investigating muscle fiber type-specific physiology and molecular mechanisms.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Accutase	Innovative Cell Technologies	AT104
Basic fibroblast growth factor	Peprotech	450-33
Bovine serum albumin	Sigma	A9418
Butorphanol tartrate	Meiji Yakuka Co., Ltd	711-12	Product name: Vetorphale injection
Chicken Embryo Extract	US Biological	C3999
Collagenase type I	Worthington	M9B2717
DAPI	Sigma-Aldrich	D9542
Fetal bovine serum	Gibco	10437-028
Glucose-free DMEM	Gibco	A14430-01
GlutaMAX	Gibco	10569
High-glucose DMEM+GlutaMAX	Gibco	10569-010
Horse serum	Life technology	16050-130
Low-protein binding filter	Millipore	SLGVR33RB
Matrigel	Corning	354230
Medetomidine hydrochloride	Kyoritsu Seiyaku Co., Ltd	8816	Product name: Dorbene Injection
Midazolam	Maruishi Pharmaceutical Co., Ltd	4987211762100	Product name: Dormicum Injection
Penicillin-streptomycin-amphotericin B suspension	Wako	161-23181
Ponceau S stain solution	Sigma-Aldrich	24-3875-5
Trypsin-EDTA (0.25%), phenol red	Gibco	25200056
Equipment
Centrifuge tubes (25 mL)	IWAKI	2362-025
Fluorescence microscope	Keyence	BZ-X810
Pasteur pipettes	IWAKI	5 3/4 IK-PAS-5P
Stereomicroscope	Leica	M165C
Sterilin dishes (100-mm)	Thermo Scientific	3020-100	Referred as Petri dish in the manuscript
Anbitodies for immunolabelling
Goat anti-mouse (H+L), Alexa Fluor 488	Thermo Fisher Scientific	A-32723	1:400
Goat anti-rabbit (H+L), Alexa Fluor 594	Thermo Fisher Scientific	A-11012	1:400
Mouse Anti-Myosin Heavy Chain	R&D Systems	MAB4470	1:400
Mouse Anti-Pax7	DSHB	AB-528428	1:100
Rabbit Anti-MyoD	Santa Cruz Biotechnology	sc-760	1:100
Anbitodies for immunoblotting
HRP-conjugated Anti-mouse IgG	Cytiva	NA931	1:1000/ horseradish peroxidase
HRP-conjugated Anti-Rabbit IgG	Cytiva	NA934	1:1000/ horseradish peroxidase
Mouse Anti-MyHC I	Sigma	M8421	1:1000/ Myosin Heavy Chain I
Mouse Anti-MyHC II	Sigma	M4276	1:1000/ Myosin Heavy Chain II
Mouse Anti-PGC-1α	Santa Cruz Biotechnology	sc-517380	1:1000/ Pparg coactivator 1 alpha
Mouse Anti-α-tubulin	Cell Signaling Technology	#3873	1:1000/ Alpha tubulin
Rabbit Anti-COXIV	Cell Signaling Technology	#4850	1:1000/ Cytochrome c oxidase IV
Rabbit Anti-GAPDH	Cell Signaling Technology	#2218	1:10000/ Glyceraldehyde-3-Phosphate Dehydrogenase
Rabbit Anti-GLUT4	Cell Signaling Technology	#2213	1:1000/ Glucose Transporter 4
Rabbit Anti-β-actin	Proteintech	20536-1-AP	1:2000/ Nonmuscle Beta-actin