Spinal Cord Injury Immunohistochemistry

To evaluate lesion size and spared tissue area, tissue was collected for immunohistochemistry (n = 7 male and female rats) at 7 dpi. Rats received an intraperitoneal pentobarbital overdose and were transcardially perfused with 0.9% saline, then 4% paraformaldehyde. Spinal cords were suspended in paraformaldehyde overnight, cyroprotected in 30% sucrose, and cryosectioned (16 μm; Gaudet et al., 2015 (link)). For immunohistochemistry, slides were incubated with 10% normal donkey serum (1 h), then with primary antibodies (overnight; mouse anti-glial fibrillary acidic protein (GFAP; 1:100; catalog #0869110, MP-Biomedicals) and rabbit anti-Iba1 (1:1000; catalog #019-19741, Wako Chemicals), then with secondary antibodies (2 h; Alexa Fluor-488 donkey anti-mouse (A-21202) and Alexa Fluor-546 donkey anti-rabbit (A-10040); both 1:500; Thermo Fisher Scientific) and DAPI (nuclear stain; catalog #D1306, Thermo Fisher Scientific). Images were captured on an Olympus IX81 Microscope and analyzed using Fiji (Schindelin et al., 2012 (link)).

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Gaudet A.D., Fonken L.K., Ayala M.T., Bateman E.M., Schleicher W.E., Smith E.J., D’Angelo H.M., Maier S.F, & Watkins L.R. (2018). Spinal Cord Injury in Rats Disrupts the Circadian System. eNeuro, 5(6), ENEURO.0328-18.2018.

Publication 2018

catalog #019-19741 Alexa Fluor-488 donkey anti-mouse (A-21202) Alexa Fluor-546 donkey anti-rabbit (A-10040); IX81 Microscope

0 9 saline Alexa fluor 488 Alexa fluor 546 Antibodies Dapi Donkey Female Gfap Immunohistochemistry Male Microscope Mouse Overdose Paraformaldehyde Pentobarbital Rabbit Rats Serum Spinal cords Stain Sucrose Tissue

Corresponding Organization :

Other organizations : The University of Texas at Austin, University of Colorado System, University of Colorado Boulder

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Variable analysis

independent variables

Tissue collection time point (7 days post-injury)

dependent variables

Lesion size
Spared tissue area

control variables

Sex (male and female rats)
Pentobarbital overdose
Transcardial perfusion with 0.9% saline and 4% paraformaldehyde
Spinal cord tissue processing (suspension in paraformaldehyde, cryoprotection in 30% sucrose, cryosectioning at 16 μm)
Immunohistochemistry (incubation with primary antibodies against GFAP and Iba1, secondary antibodies, and DAPI)

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