Immunostaining of Microglia and Neural Markers

Microglia were fixed for 20 min with 4% paraformaldehyde in 0.1 M PBS after treatment with neural debris. The cells were rinsed with PBS and incubated sequentially in: (i) blocking solution (PBS containing 5% normal goat serum and 0.1% Triton X-100), (ii) primary antibodies [MAP2 (1:1000; BioLegend, catalog no. 801801), myelin CNPase (1:1000; BioLegend, catalog no. 836404), and Iba-1 (1:500; Cell Signaling Technology, catalog no.17198S)], (iii) PBS, (iv) secondary antibody(ies) [goat anti-mouse Alexa Fluor 546 (1:1000; Invitrogen, catalog no. A11030) and goat anti-rabbit Alexa Fluor 633 (1:1000; Invitrogen, catalog no. A21071)], and (v) DAPI nuclei marker (1:1000; Sigma-Aldrich). Chamber slides were examined with a Leica confocal microscope.

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Ritzel R.M., Li Y., Jiao Y., Lei Z., Doran S.J., He J., Shahror R.A., Henry R.J., Khan R., Tan C., Liu S., Stoica B.A., Faden A.I., Szeto G., Loane D.J, & Wu J. (2023). Brain injury accelerates the onset of a reversible age-related microglial phenotype associated with inflammatory neurodegeneration. Science Advances, 9(10), eadd1101.

Publication 2023

Iba-1 Alexa Fluor 546 Alexa Fluor 633 Leica confocal microscope

After treatment Alexa fluor 546 Antibodies Antibody Cells Cnpase Confocal microscope Dapi Goat Map2 Microglia Mouse Myelin Neural Nuclei Paraformaldehyde Rabbit Serum Triton x 100

Corresponding Organization : Trinity College Dublin

Other organizations : The University of Texas Health Science Center at Houston, Howard University, Allen Institute for Immunology, University of Washington

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Variable analysis

independent variables

Treatment with neural debris

dependent variables

Microglia morphology and marker expression (MAP2, myelin CNPase, Iba-1)

control variables

Fixation duration (20 min)
Fixative concentration (4% paraformaldehyde)
Fixation buffer (0.1 M PBS)
Blocking solution (PBS containing 5% normal goat serum and 0.1% Triton X-100)
Primary antibody concentrations (MAP2 1:1000, myelin CNPase 1:1000, Iba-1 1:500)
Secondary antibody concentrations (goat anti-mouse Alexa Fluor 546 1:1000, goat anti-rabbit Alexa Fluor 633 1:1000)
DAPI nuclei marker concentration (1:1000)
Imaging method (Leica confocal microscope)

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