Computational protocol: Impaired Ca2+ release contributes to muscle weakness in a rat model of critical illness myopathy

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Protocol publication

[…] Cross-sections or longitudinal sections, 10 μm thick, were cut from the tibialis anterior (TA) muscle in a cryostat at −23 °C and placed on glass slides. Longitudinal sections were used to visualize the distribution of Na+ channels and DHPR in the sarcolemma while transverse sections were preferred to identify the distribution of the RyR and SERCA1 proteins embedded in the sarcoplasmic reticulum membrane. The tissue sections were air dried at room temperature and stored at −80 °C. The sections were fixed for 20 minutes in cold 2 % formaldehyde diluted in phosphate-buffered saline system (PBS) (Sigma-Aldrich), and then washed with 0.1 % saponin (Sigma) at pH 7.4 (referred to as PBS-Sap) for 10 minutes. The blocking step was performed with 5 % normal goat serum (Sigma-Aldrich) for 1 hour at room temperature. After washing with PBS-Sap, sections were stained with anti-ryanodine receptor 1 (RyR1; Abcam, ab2868, 1:600), anti-sarco-endoplasmic reticulum Ca2+ ATPase 1 (SERCA1; Abcam, ab109899, 1:200), anti-Na+ channel (Millipore, Catalog number 06-811, 10 μg/ml), and anti-DHPR (Abcam, ab2864, 1:600) diluted in 1 % goat serum + PBS-Sap and incubated overnight at 4 °C in a humid chamber. After washing with PBS-Sap, secondary antibodies (Alexa 488 goat anti-rabbit IgG, or Alexa 488 goat anti-mouse IgG, 1:1000, Invitrogen, Oregon, USA) diluted in 1 % goat serum + PBS-Sap were applied to the sections for 30 minutes in the dark. A negative control for each run was used (Additional file : Figure S2). Slides were mounted using the anti-fade mounting medium for fluorescence Vectashield (Vector, Burlingame, CA). Slides were examined using a Bio-Rad MRC 1024 confocal unit with a Calypso dual laser (Cobolt, Solna, Sweden) attached to a Nikon Diaphot 200 inverted microscope and a Nikon Plan Apo × 40 oil-immersion objective, N.A. 1.3 (Nikon, Tokyo, Japan). The stained sections were coded and evaluated in a descriptive manner by independent observers (MLD, JDB, VS and AJC). Images were prepared using ImageJ software (National Institutes of Health, Bethesda, MD; https://imagej.nih.gov/ij/). [...] Data are presented as mean ± SEM. Statistical analyses were performed using the SigmaPlot software (Systat Software, Inc., CA, USA). Unpaired t tests were performed to establish significant differences between groups and a p value <0.05 was considered to be statistically significant. […]

Pipeline specifications

Software tools ImageJ, SigmaPlot
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Rattus norvegicus, Homo sapiens
Diseases Muscular Diseases
Chemicals Caffeine, Ryanodine, Sodium