May 20, 2024
MyHC-IIb/Laminin Fresh Frozen
This protocol is a draft, published without a DOI.
- Griffen Wakelin1
- 1Dalhousie University
Protocol Citation: Griffen Wakelin 2024. MyHC-IIb/Laminin Fresh Frozen. protocols.io https://protocols.io/view/myhc-iib-laminin-fresh-frozen-ddyv27w6
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: May 18, 2024
Last Modified: May 20, 2024
Protocol Integer ID: 100085
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Abstract
Myosin heavy chain IIb/laminin immunofluorescence staining protocol for fresh frozen skeletal muscle.
Protocol materials
M.O.M.® (Mouse on Mouse) Blocking ReagentVector LaboratoriesCatalog #MKB-2213-1
Step 2
BF-F3 (MHC IIb)Developmental Studies Hybridoma BankCatalog #BF-F3
Step 6
Anti-Laminin antibody produced in rabbitMerck MilliporeSigma (Sigma-Aldrich)Catalog #L9393
Step 6
Alexa Fluor® 647 AffiniPure™ Goat Anti-Mouse IgM, µ chain specificJackson ImmunoResearch Laboratories, Inc.Catalog #115-605-075
Step 10
Alexa Fluor® 488 AffiniPure™ Goat Anti-Rabbit IgG (H L)Jackson ImmunoResearch Laboratories, Inc.Catalog #111-545-003
Step 10
DAPIMerck MilliporeSigma (Sigma-Aldrich)Catalog #D9542
Step 10
Staining
Staining
3h 45m
Thaw sections, dry for ~30 mins, pap, and label.
Note
The MyHC-IIb will, under no circumstances, work with fixed tissue. Heat-induced anti retrieval has been reported to work:
CITATION
however, we have not been able to get this to work ourselves.
Block M.O.M.® (Mouse on Mouse) Blocking ReagentVector LaboratoriesCatalog #MKB-2213-1 01:00:00 Room temperature
1h
Wash 1X PBS 00:05:00
5m
Wash 1X PBS 00:05:00
5m
Wash 1X PBS 00:05:00
5m
Incubate primaries in 1% BSA:
- BF-F3 (MHC IIb)Developmental Studies Hybridoma BankCatalog #BF-F3 1:10
- Anti-Laminin antibody produced in rabbitMerck MilliporeSigma (Sigma-Aldrich)Catalog #L9393 1:1000
Overnight 4 °C
Wash 1X PBS 00:05:00
5m
Wash 1X PBS 00:05:00
5m
Wash 1X PBS 00:05:00
5m
Incubate secondaries in 1% BSA:
- Alexa Fluor® 647 AffiniPure™ Goat Anti-Mouse IgM, µ chain specificJackson ImmunoResearch Laboratories, Inc.Catalog #115-605-075 1:1000
- Alexa Fluor® 488 AffiniPure™ Goat Anti-Rabbit IgG (H L)Jackson ImmunoResearch Laboratories, Inc.Catalog #111-545-003 1:1000
- DAPIMerck MilliporeSigma (Sigma-Aldrich)Catalog #D9542 1 μg/ml
02:00:00 Room temperature
2h
Wash 1X PBS 00:05:00
5m
Wash 1X PBS 00:05:00
5m
Wash 1X PBS 00:05:00
5m
Mount with aqueous mounting media. Image within 2 days.
Citations
Step 1
Esper ME, Kodippili K, Rudnicki MA.. Immunofluorescence Labeling of Skeletal Muscle in Development, Regeneration, and Disease
https://doi.org/10.1007/978-1-0716-2675-7_9