Horseradish peroxidase conjugated donkey anti mouse secondary antibody

Cells were homogenized directly into the following buffer: Tris 50 mM, NaCl 150 mM, EDTA 10 mM, and Triton-X 1% and centrifuged at 10,000g for 10 min. Protein concentrations were determined by the Bradford assay. Proteins were resolved by 16% SDS-PAGE, electrotransferred on PVDF membranes (Amersham™ Hybond™, GE Healthcare Life Science, cat. number 28906837), and blocked with 5% [v/v] nonfat dry milk/0.1% [v/v] TBS-T. The blots were probed with the following primary antibodies: mouse monoclonal anti-osteocalcin (1 : 500) (abcam-ab13420) in 5% BSA/TBS-T 0.1% and mouse monoclonal anti-beta actin (1 : 10,000) (sigma-aldrich-A5541).
Membranes were then incubated with the appropriate horseradish peroxidase-conjugated donkey anti-mouse secondary antibody (1 : 5000; Jackson), and the reaction was detected with the Western lighting Plus ECL (Perkin Elmer, Waltham, MA, USA).

The protein expression of Hsp27 was examined. In order to examine Hsp27 expression, we conducted the same experiment using another 6 hairless rats according to the design protocol (3 rats in each group). We obtained skin samples from the PI wounds by surgery biopsy from different rats at 6, 12, and 24 h after decompression for protein analysis. The samples were snap frozen immediately following resection. The tissue was homogenized in ice-cold RIPA, and then centrifuged at 12,000× g for 10 min at 4 °C. The supernatant containing the total cell protein was extracted for protein concentration assay. Samples were separated on 10% sodium dodecyl sulfate-polyacrylamide gels, and then transferred onto a nitrocellulose membrane. Each well was loaded with approximately 30 μg of soluble protein. Placental tissue, which expresses Hsp27, was used as a reference sample and positive control on each gel. The primary antibody (HSP 27, Santa Cruz) was used as 1:1000. The second antibody was horseradish peroxidase-conjugated donkey anti-mouse secondary antibody (Jackson, 1:5000). Proteins were visualized using the ECL detection system (Thermo Fisher Scientific). Bands were scanned using a GE imaging densitometer. Band densities were expressed relative to the density of the internal placental control sample included on every well by using the ImageJ software.

Two hundred young adult animals aged 48 hours from the L4 stage were picked into tris-buffered saline (TBS) with 0.1% Tween 20 buffer, washed three times, and then incubated in 1× SDS sample buffer at 50°C for 10 min. Samples were vortexed for 2 to 3 min until no visible solid material remained. Proteins were separated using SDS–polyacrylamide gel electrophoresis gradient gels (Invitrogen, NuPAGE, 4 to 12%, bis-tris, 1.0 mm, 10-well, MES SDS running buffer) and transferred to Amersham Hybond P 0.45 polyvinylidene difluoride membranes. The membrane was cut into slices and probed with mouse anti-HA (Invitrogen, 2-2.2.14) (1:1000), mouse anti-V5 (Invitrogen) (1:1000), or mouse anti–α-tubulin (Sigma-Aldrich, DM1A) (1:5000) antibodies overnight at 4°C. Horseradish peroxidase–conjugated donkey anti-mouse secondary antibody (Jackson ImmunoResearch) (1:10,000) was incubated with membranes for 1 hour at room temperature. SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) was used for detection. ImageJ mean gray value was used for quantification.