Hematoxylin & Eosin (H&E) staining

Hematoxylin & Eosin (H&E) staining of skeletal myotubes and skeletal muscle tissues

Hyun-Jun Kim¹, Da-Woon Jung¹*, and Darren R. Williams¹*

¹New Drug Targets Laboratory, School of Life Sciences, Gwangju Institute of Science and Technology, 123 Cheomdangwagi-ro, Buk-Gu, Gwangju 61005, Republic of Korea

*Correspondence: 1) darren@gist.ac.kr, 2) jung@gist.ac.kr.

Technical note: Please note that this protocol was developed for staining skeletal myotube cultures. We have not tested this protocol with other cell types. In addition, we noticed a difference in the staining intensity for human and murine myotubes: human primary myotube cultures stained more faintly for eosin in the cytoplasm compared to C2C12 myotubes. Additionally, for H&E staining of skeletal muscle, we include a protocol describing staining of the gastrocnemius muscle.

Detailed protocol for H&E staining of murine C2C12 myotubes and myotubes derived from human skeletal myoblasts (HSKM)

1. Aspirate media and wash the cells with PBS for 2 times before fixation
2. Fixation : treat 3.7 % formaldehyde solution on the cells at room temperature (RT) for 15 min
3. Wash the cells with PBS for 3 times to remove residual formaldehyde solution
4. Permeabilization : treat 0.2 % Triton X-100 solution at room temperature (RT) for 10 min 
5. Wash the cells with PBS for 3 times to remove residual permeabilization solution
6. Wash the cells with deionized water for 2 times 
7. Treat Gill No.3 Hematoxylin solution for at room temperature (RT) for 1 min
8. Wash the cells with tap water for 3 times 
9. Treat Eosin Y solution at room temperature (RT) for 45 sec 
10. Dehydration : wash the cells with 95 % and 100 % ethanol solution for 2 times on each step ( 95 % and 100 % ethanol were used to remove eosin solution ) 
11. Immerse the cells in PBS before taking pictures with microscopy

Detailed protocol for H&E staining in gastrocnemius muscle

1. Dissect out gastrocnemius muscle tissues from mice
2. Sucrose embedding : continuously incubate gastrocnemius muscle tissues in 10 %, 20 % and 30 % of sucrose solution at 4℃ for 24 h each step
3. Fixation : embed muscle tissues in 3.7 % of formaldehyde solution at 4℃ for 24 h
4. Cryo-OCT embedding : embed muscle tissues in OCT solution to make Cryo-block 
   (it’s important to make the muscle tissues upright to get accurate size of CSA)
5. Cryo-section : section the slides at 8 µm of thickness
6. Rinse the slides in deionized water for 1 min
7. Incubate the slides in chamber containing Gill No.3 Hematoxylin solution for 1 min to stain the nuclei dark blue
8. Rinse the stained slides with running tap water for 10 min
9. Incubate the slides in chamber containing Eosin Y solution for 3 min
10. Rinse the stained slides with deionized water for 2 min for 3 times to remove excess of Eosin solution
11. Dehydration : incubate the slides in 95 % and 100 % ethanol solution for 2 min for 2 times respectively
12. Incubate the slides in xylene for 2 min
13. Cover the slides with Permount and coverslip

Materials and reagents 

Results