The left gastrocnemius and soleus muscles were embedded in paraffin and immunohistochemical analysis was performed on full cross‐sections from the muscle belly. Labelling was performed using primary antibodies directed against slow‐skeletal myosin heavy chain (ab11083; Abcam), fast‐skeletal myosin heavy chain (ab91506; Abcam) and wheat germ agglutinin (W6748; Thermofisher Scientific). Images were then acquired using an epifluorescence microscope at 20× magnification (Zeiss Axio Imager M1), and analyzed with FIJI (ImageJ, NIH) to determine myofiber cross‐section area (CSA) with the help of the muscle morphometry plug‐in (by Anthony Sinadinos using Eclipse IDE); and the experimenter was blinded for treatment during picture acquisition and CSA measurement. For all measurements, the entire cross‐section of both the gastrocnemius and soleus, and consisted of ~200 myofibers per animals. The average of all myofibers within each animal was utilized for inferential statistics.
W6748
Manufactured by Thermo Fisher Scientific
Sourced in United States
The W6748 is a laboratory equipment product offered by Thermo Fisher Scientific. It is designed for general laboratory use. The core function of the W6748 is to perform a specific task required in the laboratory setting. No further details about the intended use or specific capabilities of the W6748 are provided.
Automatically generated - may contain errors
Lab products found in correlation
Ab91506, by Abcam (2 mentions)
Ab11083, by Abcam (2 mentions)
A11005, by Thermo Fisher Scientific (2 mentions)
A 11072, by Thermo Fisher Scientific (2 mentions)
Ab184630, by Abcam (2 mentions)
Sc 33735, by Santa Cruz Biotechnology (2 mentions)
Inverted sp5 aobs confocal microscope, by Leica camera (2 mentions)
Paraformaldehyde (pfa), by Merck Group (2 mentions)
Las af software, by Leica Microsystems (2 mentions)
Mab1398z, by Merck Group (1 mentions)
Dapi h 1200, by Vector Laboratories (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
5 protocols using w6748
1
Muscle Fiber Morphometry Analysis
2
Immunohistochemical Analysis of Tibialis Anterior
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Tibialis anterior cryosections were fixed in 4% paraformaldehyde for 30 min. Slides were then permeabilized in 0.5% Triton X-100 solution and then blocked in 5% BSA solution for 1 h. The slides were then incubated with CD31 antibody (Millipore MAB1398Z) overnight at 4 °C. The slides were then incubated with secondary antibodies and WGA (ThermoFisher W6748) for 4 h and mounted with VECTASHIELD mounting media with DAPI.
3
Immunofluorescence Imaging and Morphometry of Skeletal Muscle
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Soleus muscles were embedded in paraffin blocks, sectioned into 10 μm slices and triple immunofluorescence staining was performed using and anti‐slow skeletal myosin heavy chain antibody (ab11083, Abcam, Cambridge, MA, USA), anti‐fast skeletal myosin heavy chain antibody (ab91506, Abcam, Cambridge, MA, USA), and anti‐wheat germ agglutinin (W6748, Thermofisher Scientific, Waltham, MA, USA) as previously described (Mortreux et al., 2021 (link)). Stained slides were subsequently imaged at 20× using a Zeiss Axio Imager M1 epifluorescence microscope, and myofiber cross‐sectional area (CSA) was measured using the muscle morphometry plug‐in (Anthony Sinadinos using Eclipse IDE) and FIJI (FIJI, ImageJ, NIH), with experimenters being blinded during both image acquisition and image analysis. CSA data from normally loaded controls (NL R + 0) and hindlimb suspended controls (HLS R + 0) were obtained from animals whose results were the object of another publication (Mortreux et al., 2021 (link)); however, no data were duplicated.
4
Immunocytochemistry for Membrane Proteins
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Cells were grown on glass coverslips and fixed in 4% paraformaldehyde (Sigma-Aldrich) for 10 min at room temperature. Cells were blocked in 5% BSA for 1 h at room temperature. Cells were stained with the PMA1 antibody (1:100; SC-33735, Santa Cruz Biotechnology) or CA-IX antibody for 2 h (1:500, ab184630, Abcam) and washed in PBS. Cells were further incubated for 1 h with secondary anti-rabbit-Alexa Fluor 594 antibody (1:2000; A11072, Invitrogen) or anti-mouse-Alexa Fluor 594(1:2000; A11005, Invitrogen) and additionally incubated in WGA, cell membrane marker (W6748, Invitrogen) for 10 min on ice. The cells were mounted for fluorescence with DAPI (H-1200, Vector). The slides were viewed by Leica inverted SP5 AOBS confocal microscope, and micrographs were taken, and images were subsequently acquired in the Moffitt Analytic Microscopy Core Facility by using dual photomultiplier tube detectors and LAS AF software (Leica Microsystems). For detection of intracellular PMA1, cells were fixed and permeabilized with 1:1 mixture of methanol and acetone, and immunostained with PMA1 antibody for 1 h, followed by 1 h of incubation with the secondary anti-rabbit Alexa488 antibody (Molecular Probes, Invitrogen). The cells were mounted and viewed by fluorescence microscopy.
5
Immunofluorescent Staining of PMA1 and CA-IX
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Cells were grown on glass coverslips and fixed in 4% paraformaldehyde (Sigma-Aldrich) for 10 min at room temperature. Cells were blocked in 5% BSA for 1hr at room temperature. Cells were stained with the PMA1 antibody (1:100; SC-33735, Santa Cruz Biotechnology) or CA-IX antibody for 2 hours (1:500, ab184630, Abcam) and washed in PBS. Cells were further incubated for 1 h with secondary anti-rabbit-Alexa Fluor 594 antibody (1:2000; A11072, Invitrogen) or anti-mouse-Alexa Fluor 594(1:2000; A11005, Invitrogen) and additionally incubated in WGA, cell membrane marker (W6748, Invitrogen) for 10 min on ice. The cells were mounted for fluorescence with DAPI (H-1200, Vector). The slides were viewed by Leica inverted SP5 AOBS confocal microscope, and micrographs were taken, and images were subsequently acquired in the Moffitt Analytic Microscopy Core Facility by using dual photomultiplier tube detectors and LAS AF software (Leica Microsystems). For detection of intracellular PMA1, cells were fixed and permeabilized with 1:1 mixture of methanol and acetone, and immunostained with PMA1 antibody for 1h, followed by 1 hour of incubation with the secondary anti-rabbit Alexa488 antibody (Molecular Probes, Invitrogen). The cells were mounted and viewed by fluorescence microscopy.
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