Immunostaining of Dorsal Root Ganglia

DRGs were sectioned at a 20 µm thickness and processed for on-slide immunostaining. Primary antibodies used were mouse anti-CGRP (1:2000, Abcam), rabbit anti-phospho-CREB (1:1000, Cell Signaling), rabbit anti-phospho-Akt (1:500, Cell Signaling), and rabbit anti-PLC-γ (1:1500, Santa Cruz Biotechnology). The specificity of these antibodies had been carefully characterized in our previous studies.^{2 (link),13 (link)} The secondary antibodies used were Cy3- or Alexa 488-conjugated species-specific antibody. The final product of the slides was coverslipped with Citifluor (Citifluor Ltd., London) and viewed under a Zeiss fluorescent photomicroscope. Immunoreactive positive cells were counted in 6 to 10 sections randomly chosen from each ganglion and averaged as one sample. The area of section containing cells (excluding the area containing fibers) was selected using free-line tools integrated in the AxioVision measurement software (Carl Zeiss, Inc.) and was measured as mm². The number of positively stained cells was normalized against the measured area and expressed as number cells per mm². This method of quantification has been validated in our previous studies.^{13 (link)} To avoid double counting, we chose every third section for one specific antibody stained.