Does anyone know any protocol I can follow for muscle fiber-type staining for paraffin-embedded tissue?
I would suggest using these protocols from the Rudnicki lab:
Immunofluorescence Labelling of Skeletal Muscle in Development, Regeneration, and Disease. Marie E. Esper, Kasun Kodippili, and Michael A. Rudnicki. Methods Mol Biol. 2023; 2566: 113–132.
PMID: 36152246
doi: 10.1007/978-1-0716-2675-7_9
Immunohistochemistry (IHC) staining for muscle fiber typing in mouse tissue can be achieved using specific antibodies targeting different muscle fiber types. Here's a general protocol you can follow:
Materials:
1. Paraffin-embedded mouse muscle tissue sections
2. Primary antibodies against muscle fiber types (e.g., anti-Myosin heavy chain antibodies for type I, type IIa, and type IIb fibers)
3. Secondary antibodies conjugated with fluorescent dyes or enzyme-linked detection systems
4. Blocking reagents (e.g., normal serum from the species used to generate secondary antibodies)
5. Washing buffer (e.g., PBS with Tween 20)
6. Mounting medium with or without nuclear counterstain
7. Microscope slides and coverslips
Protocol:
1. Deparaffinization and rehydration: Place the tissue sections in xylene to remove paraffin, followed by a series of ethanol washes to rehydrate the tissue.
2. Antigen retrieval: Perform heat-induced antigen retrieval by heating the tissue sections in a suitable buffer (e.g., citrate buffer) in a microwave or water bath.
3. Blocking: Incubate the tissue sections with a blocking solution containing normal serum to block non-specific binding sites.
4. Primary antibody incubation: Dilute the primary antibodies against specific muscle fiber types in blocking solution according to the manufacturer's recommendations. Incubate the tissue sections with primary antibodies overnight at 4°C or for a suitable duration at room temperature.
5. Washing: Wash the tissue sections with washing buffer to remove unbound primary antibodies.
6. Secondary antibody incubation: Incubate the tissue sections with secondary antibodies conjugated with fluorescent dyes or enzyme-linked detection systems. Dilute the secondary antibodies in blocking solution according to the manufacturer's recommendations.
7. Washing: Wash the tissue sections again to remove unbound secondary antibodies.
8. Mounting: Mount the tissue sections on microscope slides using mounting medium with or without nuclear counterstain.
9. Coverslipping: Place coverslips on the mounted tissue sections.
10. Imaging: Image the stained tissue sections using a fluorescence microscope or an appropriate imaging system.
It's important to optimize the antibody concentrations, incubation times, and other experimental conditions for your specific tissue samples and antibodies. Additionally, positive and negative controls should be included in the staining protocol to ensure specificity and reliability of the results.
All the best
- Badges
- Science topic