Mouse cytokine antibody array g series

The levels of various growth factors in spinal cord tissue lysates at 24 h and seven days (D7) post SAL and DHA treatment (n = 3 mice/group/time point) were analysed using a mouse antibody array (Mouse Cytokine Antibody Array G series; RayBiotech). Lysis buffer (Raybiotech) containing proteinase inhibitor cocktail (Sigma Aldrich) was added to homogenate impact zone fragments of spinal cords, and 30 µg of each sample was incubated with the array. Incubation and washes were performed according to the manufacturer’s instructions. The fluorescence intensity for each spot was detected using an Agilent G2564B microarray scanner (Agilent Technologies), quantified by the array testing services from RayBiotech and normalized to a positive internal controls on each array allowing the array comparison. The fold change (SAL/NAÏVE; DHA/NAÏVE) in each sample was then calculated and compared. According to the manufacturer’s instructions, any ≥1.5-fold increase or ≤0.65-fold decrease in signal intensity for a single analyte between samples may be considered a measurable and significant difference in expression.

The differential level of expression of several cytokines/chemokines in serum and sciatic nerves lysates was analyzed using a mouse antibody array glass chip (Mouse Cytokine Antibody Array G series; RayBiotech) as previously described [82 (link)].
Briefly, Serum and nerve lysate samples were harvested from no-CCI (naïve), and CCI-D7 and CCI-D7 17-βEstradiol-treated female mice. Serum samples were diluted 5-fold with a blocking buffer. Lysis buffer (RayBiotech) containing proteinase inhibitor (Sigma-Aldrich) was added to homogenate sciatic nerves, protein concentration was determined, and 30 µg of each sample was added to the array. Incubation and washes were performed according to the manufacturer’s instructions. Glass chips were then incubated with biotin-conjugated primary antibody and fluorescent dye-conjugated streptavidin according to manufacturer’s instructions. Fluorescence detection was performed using an Agilent G2564B microarray scanner (Agilent Technologies) and analysis was performed using the array testing services from RayBiotech. (n = 3 each group × 3 replicates).