Muscle clearing and whole mount immunofluorescence

Muscle clearing and whole mount immunofluorescence

Bavat Bornstein^1# and Elazar Zelzer^1#

¹ Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel

# Corresponding Authors: BB bavat.bornstein@weizmann.ac.il and EZ Eli.Zelzer@weizmann.ac.il

Abstract 
This protocol describes a muscle clearing and whole-mount immunofluorescence workflow optimized for imaging intact muscle spindles. The muscle spindle is a small, specialized sensory organs embedded deep within the muscle belly. It is compatible with a variety of antibodies and fluorophores and can be adapted to visualize other small structures within muscle tissue.

Materials and Reagents

I. Reagents and Chemicals

• 40% Acrylamide solution (Bio-Rad, cat. no. 161-0140) 
• 1XPBS (Gibco, 14200-067)
• Sodium Dodecyl Sulfate (SDS) (Bio-Lab, Cat# 001981232300)
• Sodium azide (NaN₃) (Sigma, Cat# S2002)
• Histodenz (for RIMS, Millipore Sigma, D2158)
• Triton X-100 (Sigma)
• Tween-20 (Sigma)
• 2,2′-Azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (thermoinitiator, Wako, cat. no. VA-044)
• NaH₂PO₄ (monohydrate)
• Na₂HPO₄ (anhydrous)
• CAS-Block™ (Blocking solution, Invitrogen™, Cat# 008120)
• DAPI (4′,6-diamidino-2-phenylindole)
• Primary antibody (user-defined)
• Fluorophore-conjugated secondary antibody (user-defined)

II. Buffers and Solutions

A4P0 Hydrogel Solution (Prepare fresh on ice, protected from light) – 50 ml

• 5 ml 40% acrylamide solution
• 5 ml 10× PBS
• 40 ml ice-cold distilled water
• 125 mg 2,2′-Azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (thermoinitiator)

Instructions: Mix on ice, vortex until thermoinitiator dissolves. Keep cold and use immediately.

10% SDS Clearing Solution (pH 8.0, with 0.01% sodium azide) – 500 ml

• 50 g SDS
• ~480 ml 1× PBS
• 50 mg sodium azide

Instructions: In a glass bottle with a stir bar, add SDS to ~480 ml PBS. Stir until fully dissolved. Adjust pH to 8.0. Add sodium azide and fill to 500 ml. Store at room temperature.

0.1 M Phosphate Buffer (PB) – 1 liter

• 3.1 g NaH₂PO₄·H₂O
• 10.9 g Na₂HPO₄ (anhydrous)
• 0.01% sodium azide

Instructions: Dissolve salts in dH₂O. Adjust pH to 7.4. Add sodium azide and filter-sterilize. Store at RT or 4°C for up to several months.
Note: To make 0.02 M phosphate buffer for RIMS, dilute this 0.1 M buffer 1:5.

RIMS (Refractive Index Matching Solution, RI = 1.45)

• 74% (w/v) Histodenz
• 0.02 M phosphate buffer (see above)

Instructions: Mix in a sealed tube covered with foil. Shake or vortex at 37°C until fully dissolved. Store at RT, protected from light.

Wash Buffer - PBS with 0.5% Tween-20

PBST (Permeabilization Buffer) - PBS with 0.3% Triton X-100

III. Equipment

• Dissection tools (forceps, scissors, needles, dishes)
• Rotating platform/rocker
• Cold room or 4°C fridge
• 37°C incubator or shaker
• Confocal microscope (upright or inverted)

IV. Consumables

• Eppendorf tubes (1.5 ml)
• PCR tubes (0.2 or 0.5 ml)
• 5 ml tubes
• Microscope slides and coverslips
• Grease

Procedure

Step 1: Muscle Fixation and Dissection of EDL

1. Fixation:
   • Sacrifice the mouse.
   • Peel the skin and cut the hind leg.
   • Fix the leg overnight at 4°C in 4% PFA on a rotator.
2. Wash the PFA off with PBS twice (one short wash, one long wash).
3. Dissect the EDL muscles and transfer them into a fresh tube with PBS.
   For dissection details, see:
   Dissection of Mouse EDL and Soleus Muscles Protocol
4. Wash in PBS 3 times for 20 minutes at room temperature (RT) on a rotator.

Step 2: Hydrogel Embedding

Note: Use sterile tubes for all steps to avoid bacterial contamination. Embed the sample in hydrogel to maintain tissue structure during clearing.

1. Freshly prepare the A4P0 Hydrogel Solution.
2. Submerge the sample in 5 ml of ice-cold A4P0 solution, and gently shake at 4°C for 1 day.
3. Transfer the samples directly from the cold room to 37°C, standing upright without shaking for 3 hours to polymerize the hydrogel. Before moving samples to 37C, ensure the sample is at the bottom of the tube to minimize oxygen exposure, which can inhibit polymerization. After 3 hours, the hydrogel should become syrupy, not solid. 

Step 3: Lipid Removal with SDS

1. Wash the samples in a new 5 ml tube with PBS. Gently shake at RT for 5 minutes.
2. Incubate the sample in 5 ml of 10% SDS (pH 8 with 0.01% sodium azide), gently shaking at 37°C for 2–3 days, depending on muscle size.

Step 4: Staining

Note: Washing steps can be done in Eppendorf tubes; use PCR tubes for antibody staining (primary and secondary antibodies).

1. Wash the samples in wash buffer (PBS with 0.5% Tween-20) for 20 minutes at RT on a rotator.
2. Permeabilize with PBST (PBS + 0.3% Triton X-100) for 10–20 minutes at RT, depending on muscle size.
3. Wash again in wash buffer for 20 minutes at RT on a rotator.
4. Block the samples in CAS-Block™ for 4–12 hours at 37°C with gentle shaking.
5. Primary antibody incubation: Replace the blocking solution with antibody mix (1:100 in fresh blocking solution). Incubate for 3–5 days at 37°C, shaking gently.
6. Wash in wash buffer 4 times for 20 minutes each at RT on a rotator.
7. Secondary antibody incubation: Prepare secondary antibody mix (1:200) with DAPI (8 µg/ml) in blocking solution. Incubate for 2–4 days at 37°C, gently shaking.
8. Final wash: Wash in wash buffer 4 times for 20 minutes each at RT on a rotator.

Step 5: Refractive Index Matching

1. Place the samples in 500 µl RIMS solution in an Eppendorf tube.
2. Gently shake at room temperature in the dark until the sample becomes clear (usually 1–3 days).

Step 6: Embedding for Confocal Imaging

1. Place the muscle on a slide, cover it with a coverslip (with grease on the side). Add RIMS to the slide from the space between the coverslip and the slide.
2. Let the slide air dry at RT for 24 hours.
3. Image the sample using a confocal microscope.

References

1. Broellochs, A. (n.d.). Dissection of mouse EDL and Soleus muscles. protocols.io. https://dx.doi.org/10.17504/protocols.io.jcrciv6
2. Greenbaum, A., Chan, K. Y., Dobreva, T., Brown, D., Balani, D. H., Boyce, R., ... & Gradinaru, V. (2017). Bone CLARITY: Clearing, imaging, and computational analysis of osteoprogenitors within intact bone marrow. Science Translational Medicine, 9(387), eaah6518. https://doi.org/10.1126/scitranslmed.aah6518
3. Treweek, J. B., et al. (2015). Whole-body tissue stabilization and selective extractions via tissue-hydrogel hybrids for high-resolution intact circuit mapping and phenotyping. Nature Protocols, 10, 1860–1896. https://doi.org/10.1038/nprot.2015.126
4. Williams, M. P. I., Rigon, M., Straka, T., Hörner, S. J., Thiel, M., Gretz, N., ... & Rudolf, R. (2019). A novel optical tissue clearing protocol for mouse skeletal muscle to visualize endplates in their tissue context. Frontiers in Cellular Neuroscience, 13, 49. https://doi.org/10.3389/fncel.2019.00049
5. Yang, B., et al. (2014). Single-cell phenotyping within transparent intact tissue through whole-body clearing. Cell, 158, 945–958. https://doi.org/10.1016/j.cell.2014.06.044