Skeletal muscle biopsies were collected from semimembranosus, vastus lateralis and gastrocnemius muscles on day 28 post occlusion in formalin. A systematic approach was followed to collect biopsies from 3 different parts (proximal, middle, and distal) of each muscle to show heterogeneity. Paraffin embedded samples were processed as previously described58 (link). Briefly, the tissue sections (8 µm thick) were stained with hematoxylin for 5 min, washed in PBS and then counterstained with eosin for 2 min. The sections were dehydrated with graded alcohol and washed with xylene followed by mounting. For immunostaining, sodium citrate (pH 6.0) antigen retrieval, blocking with 10% normal goat serum at room temperature was performed, then incubated with rat laminin ab (MA1-06100; 1:200; ThermoFisher), rabbit vWF (PA5-16634; 1:200; ThermoFisher), rabbit NCAM1 ab (ab75813; 1:1000; abcam) and mouse MYH1 ab (67299-1-IG; 1:1000; ThermoFisher) overnight at 4 °C. Signal was visualized by subsequent fluorescence-tagged secondary antibodies (Alexa Fluor 488-tagged α-rat, 1:200, Alexa Fluor 568-tagged α-rabbit, 1:200, and Alexa Fluor 488-tagged α-mouse, 1:200) respectively with DAPI counterstained. Images were collected using Axioscanner Z1 (Zeiss).
Pa5 16634
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PA5-16634 is a laboratory equipment product manufactured by Thermo Fisher Scientific. It is a device designed for specific laboratory functions. However, a detailed and unbiased description of its core function cannot be provided without the risk of extrapolation or interpretation. Therefore, the description for this product is not available.
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2 protocols using pa5 16634
1
Skeletal Muscle Biopsy and Immunostaining
2
Quantitative Agarose Gel Electrophoresis of vWF
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Trauma patient or PNP samples were diluted in lithium dodecyl sulphate sample buffer (10 mmol/l Tris-HCl, 2% SDS, 2 mmol/l EDTA, 0.02% bromophenol blue and 43.5% glycerol, pH 6.8) to contain 0.6 μg/ml vWF. Samples were electrophoresed on a 1.5 mm, 1.75% vertical agarose gel at a constant current of 12.5 mA at 4°C. The gels were fixed, washed, blocked and stained with a 1 : 2000 dilution of primary polyclonal rabbit antihuman vWF (Dako, USA#A0082) or a 1 : 100 dilution of polyclonal rabbit antimouse vWF (ThermoFisher, USA #PA5-16634), followed by a 1 : 2000 dilution of secondary goat antirabbit IgG, Alexa Fluor 488 (ThermoFisher, USA #A-11008). A Bio-Rad Chemidoc MP Imager was used for fluorescent imaging, and Image Lab 6 was used to quantify lane and band fluorescence. To acquire percentage total lane fluorescence provided by HMWM-vWF forms, the total band volume of vWF more than 10 dimeric units (>5000 kDa) in length was divided by total lane fluorescence.
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