Twenty-four hours after treatment with intrathecal unconjugated saporin or Mac-1-saporin, mice were perfusion-fixed using phosphate buffered saline (PBS) followed by Lana’s fixative (4% paraformaldehyde and 14% picric acid in PBS). The lumbar spinal cord (L3–L5) was resected and post-fixed in Lana’s fixative for 4 hr, and then dehydrated in 30% sucrose solution overnight. Samples were then embedded in Tissue-Plus Optimal Cutting Temperature (O.C.T.) Compound (Fisher Health Care, Norwich, UK). Lumbar spinal cord sections (12 μm thick) were stained for Iba1 using an anti-Iba1 primary antibody (1:2,000, rabbit; Wako, Japan), followed by AlexaFluor 488-conjugated anti-rabbit secondary antibody (1:300; Thermo Fisher). Stained sections were mounted with DAPI-containing media (Vectashield; Vector Laboratories, Burlingame, CA, USA). ImageJ was used to analyze confocal microscope images by percent area florescence [25 (link),55 (link)] within the medial dorsal horn.
Protocol detail
Quantifying Spinal Microglia Activation
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Tissue-Plus Optimal Cutting Temperature (O.C.T.) Compound
anti-Iba1 primary antibody
AlexaFluor 488-conjugated anti-rabbit secondary antibody
Vectashield
After treatment
Anti antibody
Confocal microscope
Dapi
Dorsal horn
Fixative
Lana
Lumbar cord
Mac 1
Mice
Paraformaldehyde
Perfusion
Phosphate
Picric acid
Rabbit
Saline
Saporin
Sucrose
Tissue
Vector
Corresponding Organization :
Other organizations : The University of Texas Medical Branch at Galveston
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Variable analysis
independent variables
- Treatment with intrathecal unconjugated saporin
- Treatment with Mac-1-saporin
- Iba1 immunoreactivity (percent area fluorescence) within the medial dorsal horn of the lumbar spinal cord
- Time after treatment (24 hours)
- Perfusion fixation using phosphate buffered saline (PBS) and Lana's fixative (4% paraformaldehyde and 14% picric acid in PBS)
- Tissue processing (post-fixation in Lana's fixative, dehydration in 30% sucrose solution, embedding in Optimal Cutting Temperature (O.C.T.) Compound)
- Spinal cord region (lumbar, L3-L5)
- Tissue sectioning (12 μm thick)
- Iba1 immunostaining (primary antibody, secondary antibody, mounting with DAPI-containing media)
- Image analysis using ImageJ (percent area fluorescence in the medial dorsal horn)
- Negative control: Untreated mice
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