Raybio mouse cytokine antibody array g series

The expression levels of various cytokines/chemokines in the sciatic nerves tissue lysates of male and female mice were analyzed using a mouse antibody array glass chip (RayBio^® Mouse Cytokine Antibody Array G series; RayBiotech Inc., Norcross, GA, USA). Lysis buffer (Raybiotech, Inc) containing proteinase inhibitor (Sigma Aldrich) was added to homogenate the sciatic nerves, protein concentration was determined, and 30 µg of each sample was added to the array (at least n = 3 per group). Incubation and washes were performed according to the manufacturer’s instructions. Briefly, chip arrays were blocked at room temperature for 30 min before being incubated with 100 µL of each sample overnight at 4 °C. Glass chips were then washed and incubated with biotin-conjugated primary antibody and fluorescent dye-conjugated streptavidin according to the manufacturer’s instructions. Fluorescence detection was performed using an Agilent G2564B microarray scanner (Agilent Technologies Italy), and data extraction was performed using the array testing services from RayBiotech Norcross, GA, USA.

Levels of various cytokines/chemokines in the sera and sciatic nerves tissue lysates were analyzed using a mouse antibody array glass chip (RayBio Mouse Cytokine Antibody Array G series; RayBiotech Inc., Norcross, GA, USA) in WT mice in all experimental conditions. To obtain serum sample, blood was collected via beheading immediately following euthanasia, allowed to clot at room temperature for 30 min and then centrifuged at 3000 rpm for 15 min. Lysis buffer (Raybiotech, Inc) containing proteinase inhibitor (Sigma Aldrich) was added to sciatic nerve homogenates and 50 μg of each sample was added to the array. Incubation and washes were performed following the manufacturer’s instructions described in supplemental material. Fluorescence detection was performed using an Agilent G2564B microarray scanner (Agilent Technologies Italy) and analysis was performed using the array testing services from RayBiotech.