Computational protocol: Enhanced Cutaneous Wound Healing In Vivo by Standardized Crude Extract of Poincianella pluviosa

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Protocol publication

[…] At days 4, 7, or 10, the wounds were removed, fragmented, and homogenized with Tris buffer (50 mmol/L, pH 6.8) containing the protease inhibitors PMSF (10 mg/mL) and aprotinin (2 mg/mL) []. The samples were placed in an ultrasonic bath (3 x 15 s) and centrifuged at 4°C. Total protein (μg/mL) present in the supernatant of each sample was measured by the Bradford method [], and then diluted in a solution containing 1% SDS, 2% 2-mercaptanol, and 10% glycerol, and placed in boiling water for 5 min. Separation and packaging gels containing 10% and 4% polyacrylamide, respectively, and molecular weight standards from 6.9 to 200 kDa (Mark12 Unstained Standard) were used. GAPDH (glyceraldehyde 3-phosphate dehydrogenase) is a constitutive protein and was used as a loading control. After separation by electrophoresis in SDS-PAGE, the proteins were transferred to a nitrocellulose membrane and blocked with Tris buffer solution containing 0.2% Tween-20 (TBST, pH 7.5), and 10% milk protein for 1 h. The membrane was incubated overnight with rabbit monoclonal VEGF, COX-2, SOD-2, and GAPDH (1:250) antibody (Santa Cruz Biotechnology, Santa Cruz, CA), and washed with TBST. The membrane was revealed using a secondary antibody F(ab') 2 fragment of goat anti-mouse IgG (Santa Cruz Biotechnology) conjugated to peroxidase (1:1000) for 1 h. The blot was incubated in a chemiluminescence solution (Novex Chemiluminescent Substrates, Invitrogen) at room temperature, and was autoradiographed with a ChemiDoc XRS System (Bio-Rad). Protein levels were analyzed by densitometry (ImageJ 1.47) and normalized against the GAPDH response (100%). [...] The software Statistica 8.0 (StatSoft, Inc. 1984–2007) was used for the statistical analyses. Data are expressed as mean±standard deviation (SD) using the Mann-Whitney test, a nonparametric analysis for Western Blot; and the Tukey test, a unilateral analysis of variance (one-way ANOVA) for multiple comparisons. Significant differences were determined using p<0.05 as the significance criterion. […]

Pipeline specifications

Software tools ImageJ, Statistica
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Homo sapiens
Chemicals Superoxides