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.