FACS purification of CD11b+ microglia were performed according to previously described methods (Srinivasan et al., 2016 (link)) with some minor modifications. Briefly, nonTg and 5×FAD mice were perfused with ice-cold PBS under isoflurane anesthetization. Mice cortices and hippocampi were then dissected and incised into ice-cold DPBS (Gibco, 14190144). The brain tissue was dissociated with Accutase (Innovative Cell Technologies, NC9839010) and debris and myelin were removed with Debris Removal Solution (Miltenyi Biotec, 130–109-398). Cells were washed with DPBS followed by centrifuging at 300 ×g at 4 °C for 10 min. The cell pellet was fixed in 50% ice-cold ethanol for 15 min and centrifuged at 500 ×g at 4 °C for 10 min. The pellet was then resuspended in ice-cold DPBS and subjected to immunostaining at 4 °C for 20 min. The following antibodies and reagents were used: Alexa Flour 488-conjugated anti-NeuN (Millipore, MAB377X, 1:1000), PE-conjugated anti-GFAP (BD Biosciences, #561483, 1:50), APC-conjugated anti-CD11b (BD Biosciences, #561690, 1:250), and DAPI (Invitrogen, D21490, 5 ng/ml). The cells were washed twice with DPBS, then resuspended in DPBS. DAPI+ /singlet cells were selected for parent gating to exclude cell debris. CD11b+/NeuN− /GFAP− cell subset was sorted and collected for RNA extraction.
Pe conjugated anti gfap
Manufactured by BD
PE-conjugated anti-GFAP is a fluorescent-labeled antibody that binds to the glial fibrillary acidic protein (GFAP), a cytoskeletal protein found in astrocytes and other glial cells. This antibody can be used for the detection and analysis of GFAP-expressing cells in various applications, such as flow cytometry and immunofluorescence microscopy.
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Lab products found in correlation
Apc conjugated anti cd11b, by BD (3 mentions)
Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (2 mentions)
Alexa flour 488 conjugated anti neun, by Merck Group (2 mentions)
Accutase, by Innovative Cell Technologies (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Debris removal solution, by Miltenyi Biotec (2 mentions)
Pe conjugated anti cd31, by BioLegend (1 mentions)
Ab167453, by Abcam (1 mentions)
Ab40676, by Abcam (1 mentions)
Ab56416, by Abcam (1 mentions)
Alexa fluor secondary antibody, by Thermo Fisher Scientific (1 mentions)
Ab13970, by Abcam (1 mentions)
4 protocols using pe conjugated anti gfap
1
FACS Purification of CD11b+ Microglia
2
Characterizing Mouse Brain Pericytes
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Primary mouse brain pericytes isolated from pdgfrβcre-;α6fl/fl and pdgfrβcre+;α6fl/fl mice were incubated with an anti-α6-integrin antibody (1:100, GoH3; Abcam), to determine expression levels, for 30 min at 4°C. This was followed by incubation, for 30 min at 4°C with an appropriate FITC-conjugated secondary antibody. Unstained cells were used as a control. For characterisation of primary mouse brain pericytes, cells were washed with PBS and trypsinised at 37°C. The cell suspensions were washed in medium containing serum and centrifuged at 214 g for 3 min. Cells were washed with cold FACS buffer (1% BSA in PBS) and fixed with 4% formaldehyde for 10 min at room temperature. Cells were washed with FACS buffer and the cell suspensions were incubated with the following primary antibodies (all 1:100) for 30 min: PE-conjugated anti-CD31 (102507, Biolegend), PE-conjugated anti-Mac1 (CD11b; 101207, Biolegend), PE-conjugated anti-GFAP (561483, BD Biosciences), APC-conjugated anti-PDGFRβ (136007, Biolegend), PE–Cy7-conjugated anti-CD146 (134713, Biolegend). Cells were then washed three times in sample buffer and resuspended in a final volume of 400 ml. As a control, unstained cells were sorted by FACS. Primary mouse lung endothelial cells were incubated with PE-conjugated anti-CD31 (as above).
3
Immunocytochemical and Western Blot Analyses
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For immunocytochemical studies, the following primary antibodies were used: LC3 mouse monoclonal (M152-3/1:50; MBL), Iba1 rabbit polyclonal (019-19741/1:1,000; Wako), and Lamp1 rat polyclonal (1D4B/1:300; DHSB). Alexa Fluor secondary antibodies were from ThermoFisher (1:400). For studies involving Western blots, the following primary antibodies were used: LC3 mouse monoclonal (M186-3/1:1,000; MBL), actin (A2228/1:5,000; Sigma), p-AMPK (T172) rabbit polyclonal (2535/1:1,000; CST), AMPK rabbit polyclonal (5831/1:1,000; CST), p-TBK1 (S172) rabbit polyclonal (5483/1:1,000; CST), TBK1 rabbit polyclonal (ab40676/1:1,000; Abcam), p-p62 (S403) rat polyclonal (D343-3/1:1,000; MBL), p62 mouse monoclonal (ab56416/1:1,000; Abcam), p-S6K (T389) rabbit polyclonal (AT-7159/1:1,000; MBL), S6K rabbit polyclonal (2708/1:1,000; CST), GFP chicken polyclonal (ab13970/1:5,000; Abcam), and mCherry rabbit polyclonal (ab167453/1:1,000; Abcam). For FACS, the following antibodies were used: Alexa Flour 488–conjugated anti-NeuN (MAB377X/1:1,000; Millipore), PE-conjugated anti-GFAP (561483/1:50; BD Biosciences), and APC-conjugated anti-Cd11b (561690/1:250; BD Biosciences).
4
FACS Purification of CD11b+ Microglia
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