Immunostaining of Skeletal Muscle Fibers

The flexor digitorum brevis (FDB) muscle bundle was dissected and placed in 1ml of DMEM containing BSA plus collagenase solution pre-warmed to 37°C in a 12-well plate. After 2 hours, fibers were triturated in Ringers solution. Fibers were fixed on coverslips with 4% paraformaldehyde blocked in 1X phosphate-buffered saline (PBS) containing 10% fetal bovine serum and triton, and then immunostained at 1:100 with anti-DHPR (ma3-920, Thermo Scientific, Rockford, IL) and at 1:100 with anti-BIN1(sc-30099, Santa Cruz, Dallas, TX). The anti-EHD1 rabbit antibody was previously described [10 (link)]. Goat anti-rabbit conjugated to Alexa 488 and goat anti-mouse 594 were used at 1:2000. Slides were mounted with Vectashield with DAPI. Images were captured using a Leica SP5 II STED-CW super resolution laser scanning confocal microscope in standard mode.

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Demonbreun A.R., Swanson K.E., Rossi A.E., Deveaux H.K., Earley J.U., Allen M.V., Arya P., Bhattacharyya S., Band H., Pytel P, & McNally E.M. (2015). Eps 15 Homology Domain (EHD)-1 Remodels Transverse Tubules in Skeletal Muscle. PLoS ONE, 10(9), e0136679.

Publication 2015

SP5 II STED-CW

Anti antibody Collagenase Confocal laser scanning microscope Dapi Fetal bovine serum Goat Mouse Muscle Paraformaldehyde Phosphate Rabbit Ringers solution Saline

Corresponding Organization :

Other organizations : Northwestern University, University of Chicago, University of Nebraska Medical Center

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Variable analysis

independent variables

Collagenase solution concentration
Incubation time

dependent variables

Presence and localization of DHPR and BIN1 proteins in muscle fibers

control variables

Temperature (37°C)
Buffer (DMEM, Ringer's solution, 1X PBS)
Fixation method (4% paraformaldehyde)
Blocking solution (10% fetal bovine serum and Triton)
Antibody concentrations (1:100 for anti-DHPR and anti-BIN1, 1:2000 for secondary antibodies)
Mounting medium (Vectashield with DAPI)

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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