2.5. Immunostaining and Image Analysis

Post treatment with either IL-6 or vehicle, day in vitro (DIV) 13–15 primary DRG cultures were fixed with 4% paraformaldehyde for 10 min at room temperature (RT) followed by a triple wash with ice-cold PBS. Cells were permeabilized by 0.3% Triton X-100 for 30 min at RT. Following permeabilization, non-specific binding sites were blocked with 2% normal goat serum (NGS) for 30 min, and samples were incubated with the following primary antibodies: Anti-NeuN (1:500, ABN91, Millipore Sigma), Anti-Nav1.7 (1:200, ab65167, Abcam), and Anti-Nav1.8 (1:500, ab93616, Abcam) for 18 hours. The following day, species specific secondary, goat anti-chicken Alexa Fluor 647 (1:200, ab150171, Abcam), goat anti-mouse Alexa Fluor 546 (1:200, A-11030, Thermofisher) and goat anti-rabbit Alexa Fluor 488 (1:200, ab150077, Abcam) antibodies were incubated for 1 hour at RT. Confocal images were acquired at 20× magnification using an inverted Nikon Eclipse Ti (Nikon, Tokyo, Japan) microscope. All confocal images of stained samples were acquired with a 20× objective with a numerical aperture (NA) of 0.5 and a calculated depth of focus of 3.6 µm. To determine the percentage of Nav1.7- and Nav1.8-expressing neurons in IL-6- or vehicle-treated groups, a minimum of three regions of interest (ROIs) were selected from a total of n = 6 wells (n = 3 IL-6, n = 3 vehicle). Multi-channel images were processed in ImageJ (NIH). Briefly, a user-defined threshold was applied to all channels and neuronal somata were manually counted based on maximum intensity projections. For the normalized mean fluorescence intensity of associated Nav1.7 and Nav1.8 expression post treatment with IL-6 and NGF (or vehicle), circular ROIs were drawn across a minimum of n = 60 somata, and the signal intensity of Nav1.7 and Nav1.8 was measured using ImageJ (NIH). For all experiments, only presumptive nociceptors were selected (diameter ~ 20 µm), and cells were identified as positive for Nav1.7 and/or Nav1.8 if the mean fluorescence intensity was ≥2.5× then the average intensity of the background.