Guilherme Abbud Franco Lapin1, Bernardo Hochman, Michele Akemi Nishioka, Jessica Ruivo Maximino, Gerson Chadi, Lydia Masako Ferreira. 1. Guilherme Abbud Franco Lapin, MD, is a graduate student in the Division of Plastic Surgery, Universidade Federal de São Paulo (UNIFESP), São Paulo, SP, Brazil. Bernardo Hochman, MD, PhD, is an Affiliated Professor in the Division of Plastic Surgery, UNIFESP, São Paulo, SP, Brazil. Michele Akemi Nishioka, FT, MS, is a graduate student in the Division of Plastic Surgery, UNIFESP, São Paulo, SP, Brazil. Jessica Ruivo Maximino, PhD, is a Neuroscience Researcher, UNIFESP, São Paulo, SP, Brazil. Gerson Chadi, MD, PhD, is a Full Professor in the Experimental Neurology Department of UNIFESP, head of the Laboratory of Pathophysiology-LIM45 in the Neurosurgery Department of UNIFESP, and Coordinator of the Study Group on Neuroregeneration, also at UNIFESP, São Paulo, SP, Brazil. Lydia Masako Ferreira, MD, PhD, is a Full Professor in the Plastic Surgery Division at UNIFESP, São Paulo, SP, Brazil; Researcher IB CNPq (Brazilian National Council for Scientific and Technological Development); and Assistant Coordinator of MED III CAPES (Coordination for the Improvement of Higher Education).
Abstract
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.
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.