Dec 12, 2024
Scientific Protocol for H&E staining in Heart and Skeletal Muscle
- Jayshen Arudkumar1,2,
- Yu Chinn Joshua Chey1,2,
- Sandra Piltz1,2,
- Paul Quinton Thomas1,2,
- Fatwa Adikusuma1,2
- 1University of Adelaide;
- 2SAHMRI
External link: https://doi.org/10.1186/s44330-024-00019-y
Protocol Citation: Jayshen Arudkumar, Yu Chinn Joshua Chey, Sandra Piltz, Paul Quinton Thomas, Fatwa Adikusuma 2024. Scientific Protocol for H&E staining in Heart and Skeletal Muscle. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1qw82gr2/v1
Manuscript citation:
Arudkumar, J., Chey, Y.C.J., Piltz, S.G. et al. CRISPR-mediated generation and comprehensive phenotyping of Duchenne Muscular Dystrophy mouse models. BMC Methods 1, 19 (2024). https://doi.org/10.1186/s44330-024-00019-y
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 23, 2024
Last Modified: December 12, 2024
Protocol Integer ID: 93945
Keywords: H&E, Histology, CRISPR, DMD, Muscle, Heart, murine models of duchenne muscular dystrophy, duchenne muscular dystrophy, crispr microinjection of embryo, crispr microinjection, mouse tissue sample, mounted mouse tissue sample, crispr, broader landscape of genetic disorder investigation, skeletal muscle paper, cas9 gene, molecular testing at the dna, tissue sample, genetic disorder investigation, molecular testing, serum creatine kinase, using mouse model, murine model, generation of diverse animal model, permanent modifications to dna
Disclaimer
These protocols are for research purposes only.
Abstract
Paper abstract: CRISPR-Cas9 gene-editing technology has revolutionised the creation of precise and permanent modifications to DNA, enabling the generation of diverse animal models for investigating potential treatments. Here, we provide a protocol for the use of CRISPR-Cas9 to create murine models of Duchenne Muscular Dystrophy (DMD) along with a step-by-step guide for their phenotypic and molecular characterisation. The experimental procedures include CRISPR microinjection of embryos, molecular testing at the DNA, RNA, and protein levels, forelimb grip strength testing, immunostaining and serum creatine kinase (CK) testing. We further provide suggestions for analysis and interpretation of the generated data, as well as the limitations of our approach. These protocols are designed for researchers who intend on generating and using mouse models to study DMD as well as those seeking a detailed framework of phenotyping to contribute to the broader landscape of genetic disorder investigations.
Protocol summary: The following protocol describes H&E staining of snap frozen cork-mounted mouse tissue samples. These include sections from the heart, tibialis anterior (TA), triceps and quadriceps.
Image Attribution
BioRender was used to generate figures for this manuscript.
Materials
• Lillie Mayer’s Haematoxylin (0.5%)
• Tap water
• 0.1% Eosin
• Coverslip
• DPX mountant (Sigma-Aldrich)
• Slide staining tray, rack (ProSci Tech)
Troubleshooting
Safety warnings
Wear proper PPE (gloves, safety goggles, enclosed shoes and lab coat) and prepare solvents in a chemical fume hood. Dispose used solvents or waste material in an appropriate biohazard waste containers.
Ethics statement
Animal work described in this manuscript has been approved and conducted under the oversight of the Animal Ethics Committee of South Australian Health and Medical Research Institute (SAHMRI) and The University of Adelaide.
Preparation of Slides
Allow the slides to air dry for 5 minutes to eliminate any residual moisture.
Haematoxylin & Eosin Staining
Immerse the slide in the following reagents order:
Haematoxylin for 5 min. Drain the excess of the hematoxylin by tapping the slide tray on paper towels
Place slide staining tray into a container and run through tap water for 2-minutes
Eosin stain solution for 3 min. Drain the excess eosin by tapping the slide tray on paper towels
Dehydration Steps
Dip slide tray twice into 70% ethanol
Dip slide tray four times into 95% ethanol
100% ethanol for 1 min and
repeat in clean 100% ethanol for 1 min.
Xylene for 1 min and
repeat in clean xylenes for 1 min. Drain the excess xylenes by tapping the slide on paper towels.
Mounting and Imaging
Add one drop of DPX mounting medium to the tissue section and cover with an appropriate-sized coverslip. Drain any excess mounting media. Allow the slide to air dry in a fume hood.
Visualise the stained tissue under a light microscope using bright-field illumination.