A Research Design for the Quantification of the Neuropeptides Substance P and Calcitonin Gene-Related Peptide in Rat Skin Using Western Blot Analysis
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2015-06-01
Autores
Lapin, Guilherme Abbud Franco 
Hochman, Bernardo
Nishioka, Michele Akemi
Maximino, Jessica Ruivo
Chadi, Gerson
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OBJECTIVE: To describe and standardize a protocol that overcomes the technical limitations of Western blot (WB) analysis in the quantification of the neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) following nociceptive stimuli in rat skin.DESIGN: Male Wistar rats (Rattus norvegicus albinus) weighing 250 to 350 g were used in this study. Elements of WB analysis were adapted by using specific manipulation of samples, repeated cycles of freezing and thawing, more thorough maceration, and a more potent homogenizer; increasing lytic reagents; promoting greater inhibition of protease activity; and using polyvinylidene fluoride membranes as transfer means for skin-specific protein. Other changes were also made to adapt the WB analysis to a rat model.SETTING: University research center.MAIN OUTCOME MEASURE: Western blot analysis adapted to a rat model.RESULTS: This research design has proven effective in collecting and preparing skin samples to quantify SP and CGRP using WB analysis in rat skin.CONCLUSION: This study described a research design that uses WB analysis as a reproducible, technically accessible, and cost-effective method for the quantification of SP and CGRP in rat skin that overcomes technical biases.
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Advances In Skin & Wound Care. Philadelphia: Lippincott Williams & Wilkins, v. 28, n. 6, p. 259-265, 2015.