Protocol detail

Spinal Protein Expression Analysis

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Western blot analyses were performed on L4/5 spinal ipsilateral dorsal quadrants, as previously described.^{23 (link),26 (link)} Primary antibodies and dilution ratios used were: rabbit P2X4R 1:400 (Alomone Labs, Jerusalem, Israel), rabbit p38 1:1000 (Cell Signaling, Danvers, MA), rabbit phospho-p38 1:1000 (Cell Signaling), rabbit p65/NFκB 1:500 (Merck Millipore, Billerica, MA), rabbit NLRP3 1:500 (LifeSpan Biosciences, Seattle, WA), guinea pig GLT-1 1:5000 (Merck Millipore), goat Iba1 1:1000 (Abcam, Cambridge, MA), rabbit CD11b 1:500 (Abcam), rabbit ATF3 1:1000 (Santa Cruz Biotechnology, Dallas, TX). Mouse β actin 1:100,000 (Sigma) was used against loading control protein. Secondary antibodies used were: Goat anti-mouse IRDye 680RD 1:15,000 (LI-COR Biosciences, Lincoln, NE), goat anti-rabbit IRDye 800CW 1:15,000 (LI-COR Biosciences), donkey anti-guinea pig IRDye 800CW 1:15,000 (LI-COR Biosciences), and donkey anti-goat IRDye 800CW 1:15,000 (LI-COR Biosciences). Bands were quantified using Image Studio (LI-COR Biosciences).

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Grace P.M., Fabisiak T.J., Green-Fulgham S.M., Anderson N.D., Strand K.A., Kwilasz A.J., Galer E.L., Walker F.R., Greenwood B.N., Maier S.F., Fleshner M, & Watkins L.R. (2016). Prior voluntary wheel running attenuates neuropathic pain. Pain, 157(9), 2012-2023.

Publication 2016

rabbit p38 goat Iba1 Mouse β actin Goat anti-mouse IRDye 680RD goat anti-rabbit IRDye 800CW donkey anti-guinea pig IRDye 800CW donkey anti-goat IRDye 800CW Image Studio

Actin Antibodies Cd11b Dilution Donkey Goat Guinea pig Irdye 800cw Mouse Nfκb Protein Rabbit Western blot analyses

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Protein expression levels of P2X4R, p38, phospho-p38, p65/NFκB, NLRP3, GLT-1, Iba1, CD11b, and ATF3

control variables

Mouse β actin protein expression used as loading control

controls

Positive controls: None mentioned
Negative controls: None mentioned

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