Immunohistochemical Identification of Muscle Fiber Types in Mice Tibialis Anterior Sections

Materials and Reagents

1. High Profile microtome blades 818 (Leica Biosystems, Germany, catalog number: 14035838926)
2. Microscope glass slides (Globe Scientific, USA, catalog number: GS-1308)
3. Camel hair brushes
4. Syringe plunger
5. 1.5 ml microfuge tubes (USA Scientific, catalog number: 1615-5500)
6. Liquid blocker Pap pen (Sigma Aldrich, catalog number: Z377821)
7. Freshly dissected mouse hind limb tibialis anterior muscle
8. Myh fast 2A antibody (Developmental Studies Hybridoma Bank, catalog number: 2F7)
9. Myh fast 2B antibody (Developmental Studies Hybridoma Bank, Iowa City, IA, catalog number: 10F5)
10. Myh slow (type1) antibody (Developmental Studies Hybridoma Bank, Iowa City, IA, catalog number: A4.840)
11. Biotinylated sheep anti-mouse IgG antibody (Abcam, catalog number: ab6807)
12. Tissue-Tek Optimal cutting temperature (OCT) compound (Sakura Finetek, USA, catalog number: 4583)
13. Isopentane (Alfa Aesar, catalog number: 19387, CAS number: 78-78-4)
14. Liquid Nitrogen
15. Bovine serum albumin (BSA) (Roche, Switzerland, catalog number: 10711454001)
16. Normal sheep serum (NSS) (Sigma-Aldrich, catalog number: S22)
17. Tween 20 (RPI Corp., USA, catalog number: P20370, CAS number: 9005-64-5)
18. 4',6-diamidino-2-phenylindole Dihydrochloride (DAPI) (Invitrogen, catalog number: D1306)
19. ProLong Gold Anti-fade solution (Invitrogen, catalog number: P10144)
20. Streptavidin Alexa Fluor 488 conjugate (Invitrogen, catalog number: S-11223)
21. Sodium phosphate, monobasic 
22. Sodium phosphate, dibasic 
23. 37% Formaldehyde (Sigma-Aldrich, catalog number: 252549, CAS number: 50-00-0)
24. Milli-Q water 
25. Blocking mixture (see Recipes)
26. Diluent (see Recipes)
27. 10% Buffered formalin (see Recipes)
28. 0.2 μg/ml DAPI in 1x PBS (see Recipes)
29. 1x Phosphate buffered saline (PBS), pH 7.4 (see Recipes)
    

Equipment

1. Rotary Cryostat Microtome (CM1950, Leica Biosystems, Germany)
2. Orbital shaker (Boeco, Germany, Ref: BOE 8069200)
3. Leica CTR 6500 microscope equipped with Leica DFC 310 FX camera
4. Cryostat
   

Software

1. Image J (Fiji, https://imagej.nih.gov/ij/)

Procedure

1. Sample preparation
   1. Dissect hind limb tibialis anterior muscle and remove the surrounding connective tissue.
   2. Holding the tendon side of the muscle, place the tissue on the flat side of the syringe plunger.
   3. Cover the tissue with OCT compound and immediately snap-freeze the tissue by immersing in isopentane cooled with liquid nitrogen for 30-40 s.
   4. Store the frozen tissues at -80 °C for future use.
   5. Cut 10 μm transverse serial sections at the mid-belly region of the muscle in a cryostat maintained at -20 °C.
   6. Support the sections while cryosectioning with a brush and pick the sections by placing a glass slide (kept at room temperature) in very close proximity to the sections.
      
      
      
2. Immunostaining of the muscle sections
   1. Draw a perimeter around the tissue section with a liquid blocker pen. This helps in staining multiple tissue sections on a glass slide and also reduces the volume of diluent required. 
   2. Add freshly prepared blocking solution (minimum of 200 μl/section) onto sections to completely cover it and incubate at room temperature for an hour on low speed orbital shaker.
   3. Prepare 1:25 dilutions of Myh fast 2A, Myh fast 2B and Myh slow (type I) primary antibody in separate 1.5 ml microcentrifuge tubes with the diluent and incubate the sections overnight at 4 °C on a low speed orbital shaker.
   4. Wash the sections with 1x PBS 3 times for 5 min each to remove the non-specific antibody bound to the sections.
   5. Fix the primary antibody staining with 10% buffered formalin for 5 min and then wash three times with 1x PBS for five min each. It is recommended (specifically for DSHB MyHC primary antibodies) that the fixation should not be performed before primary antibody incubation, as this would disrupt the efficient binding of primary antibodies to different MyHC in the frozen sections. Hence fixation is performed after primary antibody incubation step.
   6. Incubate the sections with 1:200 dilution of biotinylated sheep anti-mouse IgG secondary antibody made in diluent for one hour at room temperature with shaking.
   7. Wash the sections with 1x PBS 3 times for 5 min each and incubate with 1:400 dilution of streptavidin Alexa Fluor 488 conjugate tertiary antibody in 0.2% BSA and 0.1% PBS-Tween 20 (0.1% Tween 20 made in 1x PBS) for one hour at room temperature in the dark.
   8. Wash the sections with 1x PBS twice for 5 min each.
   9. Stain the myonuclei with DAPI made in 1x PBS diluted at 1:5,000 for 5 min and rinse thoroughly with 1x PBS.
   10. Air-dry the slides completely to mount with ProLong Gold Anti-fade mountant solution and allow it to dry overnight in the dark.
   11. Visualize using Leica CTR 6500 fluorescent microscope, equipped with the Leica DFC 310 FX camera under the FITC channel with wavelengths of excitation at 488 nm and emission at 536 nm.

Data analysis

To quantify the number of positive myofibers of each antibody used (Myh fast 2A, Myh fast 2B and Myh slow (type I), the images are imported into ImageJ (Fiji) and brightly stained muscle fibers from each antibody stained sections are counted. An example of this can be found in Figure 7 of the original research article Rao et al., 2019, which is again mentioned in this manuscript as Figure 1. The values were expressed as mean muscle fiber number ± SE of two animals. Level of significance was calculated using two-tailed Student’s t-test in comparison to wild-type muscle (***P < 0.001) (Rao et al., 2019).


Figure 1. Showing the different types of MyHC (type I, type IIa, and type IIb) staining on Akirin1 knockout (Aki1^-/-) and wild‐type (WT) tibialis anterior muscle sections (Adapted from Rao et al., 2019).

Recipes

1. Blocking mixture
   0.5% Triton X-100
   0.2% BSA
   10% NSS
   1x PBS 
2. Diluent
   0.2% BSA
   5% NSS
   0.1% Tween 20 (made in 1x PBS)
3. 10% Buffered formalin

   Sodium phosphate, monobasic	4 g
   Sodium phosphate, dibasic	6.5 g
   37% Formaldehyde	100 ml
   Milli-Q water	900 ml

4. 0.2 μg/ml DAPI in 1x PBS
   
   1. Make stock concentration of 1 mg/ml DAPI 
   2. Take 2 μl of the stock and make up to 10 ml with 1x PBS 
5. 1x Phosphate buffered saline (PBS), pH 7.4
   

   Distilled water	800 ml
   Sodium Chloride	8 g
   Potassium Chloride	0.2 g
   Sodium phosphate, dibasic	1.44 g
   Potassium dihydrogen phosphate	0.24 g

   Adjust the pH to 7.4 with HCl
   Make up the volume to 1 L
   

Acknowledgments

This work was supported by the funding agency: BMRC 07/1/21/19/ 521, NMRC/EDG/0026/2008, and Startup grant. The authors acknowledge National University of Singapore for the scholarship provided to Vanitha V. Rao throughout the course of her PhD. The authors acknowledge the original research article Rao et al., 2019 from which this protocol was first discussed.

Competing interests

The authors declare no financial and non-financial competing interests.

Ethics

The animals used in the original research article were housed at the Nanyang Technological University Animal House, Singapore. All experiments were performed as per the approved protocols of Institutional Animal Ethics Committee (Singapore) with approval number ARF‐SBS/NIE-A0270.

References

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