As previously described by our team25 (link), cells were washed then fixed in 1.5% paraformaldehyde for 20 min at room temperature. Cells were re-rinsed and permeabilized using a 90% methanol/PBS solution. This step was realized on ice for a total period of 20 min. An additional wash cycle was performed, and cells were labeled for 30 min using the following fluorescence conjugated primary antibodies: pSTAT1 (BD Biosciences, AB_1645373), pSTAT3 (BD Biosciences, AB_647232), pSTAT5 (BD Biosciences, AB_399858), pERK1/2 (BD Biosciences, AB_399857), phosphorylated p38 (BD Biosciences, AB_399856) and Phospho NF-κB p65 (Thermofisher, AB_2572751). Cells were washed for the last time and flow cytometry analysis using the BD Accuri C6 Plus flow cytometer allowed estimation of ERK1/2, NF-κB, STAT1, STAT3, STAT5 and P38 phosphorylation levels. The histogram subtraction technique was applied based on Overton subtraction and thus the overlay of the histograms of interest. The analysis was then realized by the FCS express De Novo software42 (link).Three biological replicates were carried out.
Phospho nf κb p65
Manufactured by Thermo Fisher Scientific
The Phospho NF-κB p65 is a laboratory instrument designed to detect and quantify the phosphorylated form of the NF-κB p65 subunit. It is used to study the activation of the NF-κB signaling pathway, which is involved in a variety of cellular processes, including immune response, inflammation, and cell survival.
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Lab products found in correlation
Phosphorylated p38, by BD (1 mentions)
Perk1 2, by BD (1 mentions)
Pstat1, by BD (1 mentions)
Pstat3, by BD (1 mentions)
Erk1 2, by BD (1 mentions)
Pstat5, by BD (1 mentions)
Accuri c6 plus flow cytometer, by BD (1 mentions)
Alexa fluor 647 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Glial fibrillary acidic protein (gfap), by Thermo Fisher Scientific (1 mentions)
Phospho p38, by Thermo Fisher Scientific (1 mentions)
Prolong diamond, by Thermo Fisher Scientific (1 mentions)
Superblock, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Pbs t, by Merck Group (1 mentions)
2 protocols using phospho nf κb p65
1
Flow Cytometric Analysis of Signaling Pathways
2
Immunofluorescence Analysis of Neuroinflammation
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Frozen brain tissue was cryosectioned at 12 μm, mounted onto TruBond 380 slides (Fisher-Scientific), fixed in 10% buffered formalin (Fisher-Scientific) for 10 minutes, and washed 3x in PBS for 5 minutes. Slides were blocked using SuperBlock (Thermo-Fisher) containing 0.3% Triton X-100 for 1 hour at room temperature. Samples were immunostained with primary antibodies overnight at 4°C: (Cell Signaling Technology) phospho-NF-κB P65 (catalog no. 3033T), phospho-ERK1/2 (catalog no. 4370S); (Thermo-Fisher) CD45 (catalog no. 14–0451–82 and catalog no. MA5–17687), phospho-P38 (catalog no. MA5–15177), KI67 (catalog no. PA5–19462), phospho-IRAK-1 (catalog no. PA5–105752), phospho-IRAK-4 (catalog no. BS-10208R), GFAP (catalog no. PA1–10004), and CD19 (catalog no. 14–0193–82). Slides were washed 3x in PBS containing 0.1% Tween-20 (PBS-T, Sigma-Aldrich) for 5 minutes. Fluorescently-labeled secondary antibodies were added for 1 hour at room temperature: (Thermo-Fisher) goat anti-rabbit AlexaFluor 647 (catalog no. A-21244), goat anti-chicken AlexaFluor 488 (catalog no. A-11039), goat anti-rat AlexaFluor 568 (catalog no. A-11077), and DAPI (Sigma-Aldrich). Slides were washed 3x in 1 x PBS-T for 5 minutes each and coverslips mounted using ProLong Diamond (Thermo-Fisher). Images were acquired using a Nikon A1RMP confocal microscope and analyzed using Imaris x64 v9.7.0 imaging software.
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