Immunohistochemical Quantification of Spinal Cord Astrocytes and Microglia

Spinal cord tissues (L4-L5) were routinely paraffin-embedded, sectioned, dewaxed and hydrated before antigen repair with proteinase K (0.2 mg/mL) and 10 m/Vl sodium citrate solution (pH 6.0). The sections were closed with 2% bovine serum albumin (BSA) for 30 minutes at RT and then kept with rat anti-GFAP monoclonal antibody (1:500; Millipore, USA) and rabbit anti-lbal monoclonal antibody (1:300; Wako Pure Chemical Industries, Ltd Japan), respectively, for 24 hours at 4°C. Next, they were maintained with the HRP-labeled secondary antibody for 2 hours and developed with diaminobiphenyl (DAB) amine solution. The number of GFAP-positive astrocytes and Ibal-positive microglia in spinal cord tissues was estimated by utilizing the Image-Pro Plus image analysis software system (Media Cybernetics, USA) [25 (link)].

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Yu Y., Wang M., Yu X., Yan Y., Yu B, & Zhang D. (2022). Targeting Forkhead box O1-aquaporin 5 axis mitigates neuropathic pain in a CCI rat model through inhibiting astrocytic and microglial activation. Bioengineered, 13(4), 8567-8580.

Publication 2022

Image-Pro Plus image analysis software system

Amine Anti antibody Anti gfap antibody Antibody Antigen Astrocytes Bovine serum albumin Gfap Microglia Monoclonal antibody Paraffin Proteinase k Rabbit Sodium citrate Spinal cord Tissues

Corresponding Organization :

Other organizations : Nanchang University, First Affiliated Hospital of Nanchang University, University of Chinese Academy of Sciences

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Variable analysis

independent variables

Proteinase K concentration (0.2 mg/mL)
Sodium citrate solution (pH 6.0)

dependent variables

Number of GFAP-positive astrocytes
Number of Iba1-positive microglia

control variables

Spinal cord tissues (L4-L5)
Paraffin-embedding, sectioning, dewaxing, and hydration
Antigen repair with proteinase K and sodium citrate solution
Blocking with 2% bovine serum albumin (BSA) for 30 minutes at room temperature
Incubation with primary antibodies (rat anti-GFAP and rabbit anti-Iba1) for 24 hours at 4°C
Incubation with HRP-labeled secondary antibody for 2 hours
Development with diaminobiphenyl (DAB) amine solution

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