Journal: STAR Protocols

Article Title: Protocol for enhancing RNA yield and quality from single nuclei isolated from mouse brain tissue

doi: 10.1016/j.xpro.2024.103495

Figure Lengend Snippet: Single nuclei sorting and RNA quality assessment (A) Schematic overview of the FACS sorting process. (B) Representative FACS plot showing the separation of nuclei populations based on the expression of EAAT1 and NeuN markers, with distinct clusters identified for each marker. (C) Bioanalyzer gel depicting RNA extracted from EAAT1 + and NeuN + nuclei fixed in glyoxal or PFA solutions. (D) Electropherogram displaying 18S and 28S rRNA peaks from EAAT1 + and NeuN + nuclei fixed in glyoxal or PFA solutions. (E) Analysis of RNA concentration, RIN, and DV200 scores from EAAT1 + and NeuN + nuclei fixed in glyoxal or PFA solutions (N = 3–4 per group). Statistical analysis was performed using an unpaired, two-tailed Student’s t-test.

Article Snippet: Mouse monoclonal anti-EAAT1 (1:100 dilution) , Santa Cruz Biotechnology , Cat# SC-515839, not in RRID.

Techniques: Expressing, Marker, Concentration Assay, Two Tailed Test

Journal: STAR Protocols

Article Title: Protocol for enhancing RNA yield and quality from single nuclei isolated from mouse brain tissue

doi: 10.1016/j.xpro.2024.103495

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-EAAT1 (1:100 dilution) , Santa Cruz Biotechnology , Cat# SC-515839, not in RRID.

Techniques: Recombinant, Electron Microscopy, Staining, Amplification, SYBR Green Assay, Software

Journal: International Journal of Molecular Sciences

Article Title: Neuroprotective Potential of L-Glutamate Transporters in Human Induced Pluripotent Stem Cell-Derived Neural Cells against Excitotoxicity

doi: 10.3390/ijms241612605

Figure Lengend Snippet: Expression of EAAT1 and EAAT2 in hiPSC-derived neural cells. ( A ) The significant increase in the mRNA expression levels of EAAT1 ( a1 ) and EAAT2 ( a2 ) along with culture days was confirmed by qRT-PCR ( n = 3). ** p < 0.01 vs. DIV 0 group, Tukey’s test following ANOVA. Data are expressed as the means ± standard deviations. Similar results were obtained in three independent experiments. ( B ) Representative immunoblot at 14 and 63 DIV ( b1 ). The expression level of each marker was normalized to 14 DIV. The expression levels of EAAT1 ( b2 ) and EAAT2 ( b3 ) protein tended to increase with culture days. Similar results were obtained in four independent experiments. ( C ) Identification of cell types expressed EAAT1 and EAAT2 at 63 DIV. We used the following cell markers: GFAP+Nestin+ for radial glial cells, GFAP+S100β+ for astrocytes, and HuC/D+ or MAP2+ for neurons. ( c1 ) EAAT1 was localized in radial glial cells (top) and astrocytes (bottom). ( c2 ) EAAT2 was localized in radial glial cells (top), astrocytes (middle), and neurons (bottom). Scale bar, 100 µm. Similar results were obtained in three independent experiments.

Article Snippet: Mouse anti-HuC/D monoclonal antibody (1:100, A21271, Thermo Fisher Scientific), chicken anti-GFAP polyclonal antibody (1:400, ab4674, Abcam), rabbit anti-S100β polyclonal antibody (1:500, ab52642, Abcam), goat anti-Vglut2 polyclonal antibody (1:500, Go-Af310-1, Frontier Institute, Hokkaido, Japan), rabbit anti-Syn1 polyclonal antibody (1:1000, AB1543, Chemicon), chicken anti-MAP2 polyclonal antibody (1:5000, ab5392, Abcam), mouse anti-PSD95 monoclonal antibody (1:500, 7E3-1B8, Thermo Fisher Scientific), mouse anti-EAAT1 monoclonal antibody (1:200, ab49643, Abcam), rabbit anti-Nestin polyclonal antibody (1:200, ABD69, Millipore), or guineapig anti-EAAT2 polyclonal antibody (1:1000, AB1783, Chemicon) were used.

Techniques: Expressing, Derivative Assay, Quantitative RT-PCR, Western Blot, Marker

Journal: International Journal of Molecular Sciences

Article Title: Neuroprotective Potential of L-Glutamate Transporters in Human Induced Pluripotent Stem Cell-Derived Neural Cells against Excitotoxicity

doi: 10.3390/ijms241612605

Figure Lengend Snippet: Inhibition of EAATs increases the extracellular L-Glu concentration and leads to excitotoxicity at 63 DIV. ( A ) The effect of L-Glu alone on cell viability at 14 and 63 DIV ( n = 6). Cell viability was assessed using the MTT reduction assay. At 14 ( a1 ) and 63 ( a2 ) DIV, the application of L-Glu at 100 μM for 24 h did not change MTT reductions compared with the application of DMSO at 0.4% (Cont), which was used as a vehicle. Unpaired t -test. Data are expressed as the means ± standard deviations. Similar results were obtained in three independent experiments. ( B ) The effects of TFB-TBOA (TFB, nonspecific EAAT inhibitor, 30 nM) when coapplied with L-Glu on cell viability at 14 and 63 DIV ( n = 4–6). At 14 DIV, TFB caused no effect on MTT reductions ( b1 ). On the other hand, at 63 DIV, TFB significantly decreased MTT reductions, and AP5 (NMDAR antagonist, 100 μM) blocked the decrease in MTT reductions by TFB ( b2 ). Similar results were obtained in three independent experiments. ** p < 0.01 vs. L-Glu(+)TFB(−)AP5(−) group by Tukey’s test following ANOVA. ## p < 0.01 vs. L-Glu(+)TFB(+)AP5(−) group by Tukey’s test following ANOVA. Data are expressed as the means ± standard deviations. Similar results were obtained in three independent experiments. ( C ) Identification of the contribution of specific EAATs to the decrease in exogenously applied L-Glu. ( c1 ) Change in the concentration of L-Glu in the medium ([L-Glu] out ) after L-Glu (100 μM) was applied at 63 DIV ( n = 3). [L-Glu] out was nearly zero at 60 min. Data are expressed as the means ± standard deviations. Similar results were obtained in three independent experiments. ( c2 ) The effects of EAAT inhibitors on L-Glu uptake at 63 DIV ( n = 5–7). The effects of EAAT inhibitors on the decrease in [L-Glu] out were assessed at 30 min, which was the time for the 50% decrease in [L-Glu] out from C(c1). The percentage inhibition of EAAT inhibitors on L-Glu uptake activity was calculated as 100% of the decrease in extracellular L-Glu concentration in the absence of EAAT inhibitors. UCPH-101 (UCPH, EAAT1 selective EAAT1 inhibitor, 100 μM), dihydrokainic acid (DHK, a competitive EAAT2 inhibitor, 300 μM), WAY213613 (WAY, 10 μM), or TFB inhibited L-Glu uptake. Data are expressed as the means ± standard deviations. ( D ) Inverse correlation between the strength of L-Glu uptake inhibition and the cell viability caused by EAAT inhibitors. The Pearson’s correlation coefficient (PCC) = −0.7110. EAAT inhibitors are shown as follows: UCPH: blue dot; DHK: green dot, WAY: pink dot, and TFB: red dot.

Article Snippet: Mouse anti-HuC/D monoclonal antibody (1:100, A21271, Thermo Fisher Scientific), chicken anti-GFAP polyclonal antibody (1:400, ab4674, Abcam), rabbit anti-S100β polyclonal antibody (1:500, ab52642, Abcam), goat anti-Vglut2 polyclonal antibody (1:500, Go-Af310-1, Frontier Institute, Hokkaido, Japan), rabbit anti-Syn1 polyclonal antibody (1:1000, AB1543, Chemicon), chicken anti-MAP2 polyclonal antibody (1:5000, ab5392, Abcam), mouse anti-PSD95 monoclonal antibody (1:500, 7E3-1B8, Thermo Fisher Scientific), mouse anti-EAAT1 monoclonal antibody (1:200, ab49643, Abcam), rabbit anti-Nestin polyclonal antibody (1:200, ABD69, Millipore), or guineapig anti-EAAT2 polyclonal antibody (1:1000, AB1783, Chemicon) were used.

Techniques: Inhibition, Concentration Assay, MTT Reduction Assay, Activity Assay

Journal: eLife

Article Title: DRAXIN regulates interhemispheric fissure remodelling to influence the extent of corpus callosum formation

doi: 10.7554/eLife.61618

Figure Lengend Snippet: Schema of interhemispheric midline at E12 ( A ), E15 ( D dorsal; G ventral) and E17 ( I ). In situ hybridisation for Draxin mRNA (white or green), with immunohistochemistry for astroglial marker, GLAST (red), and leptomeninges and IHF marker, Laminin (LAM; magenta) in E12 ( B ), E15 ( E ), and E17 ( J ) wildtype CD1 mid-horizontal telencephalic midline tissue sections. Yellow arrowheads indicate Draxin -positive/GLAST-positive glia. Open red arrowheads indicate lack of Draxin mRNA within the IHF (yellow outlined). Immunohistochemistry for DRAXIN (white or green), GLAST (red or magenta), and LAM (magenta) in E12 ( C ), E15 ( F ), and E17 ( K ) wildtype CD1 mid-horizontal telencephalic midline tissue sections. ( H ) DRAXIN (white or green), axonal marker GAP43 (red), and LAM (magenta) in E15 ventral telencephalic midline tissue sections. Yellow arrowheads indicate regions of DRAXIN protein on GLAST-positive glial fibres ( C, F, K ) or DRAXIN protein on GAP43-positive axons ( H’ ). White arrowheads indicate DRAXIN protein within the IHF and on the basement membrane of the IHF. BM: basement membrane; CCx: cingulate cortex; IGG: indusium griseum glia; LM: leptomeninges; MZGp: midline zipper glia progenitors; Se: septum; Th: telencephalic hinge; 3V: third ventricle.

Article Snippet: Antibody , Mouse monoclonal anti-Glast (EAAT1) , Abcam , Ab49643, RRID: AB_869830 , ‘(1:500)’.

Techniques: In Situ, Hybridization, Immunohistochemistry, Marker

Journal: eLife

Article Title: DRAXIN regulates interhemispheric fissure remodelling to influence the extent of corpus callosum formation

doi: 10.7554/eLife.61618

Figure Lengend Snippet: Wildtype C57 and BTBR pregnant mice were injected with ethynyl deoxyuridine (EdU) every 24 hr from E12 and collected 24 hr later ( D ). Immunohistochemistry for GLAST (white), EdU (green), and cell cycle marker, KI67 (red) on E13 ( A ), E14 ( B ), and E15 ( C ) wildtype C57 and BTBR horizontal brain sections of the telencephalic hinge and interhemispheric fissure (IHF) base. ( E ) To determine whether the litters were age-matched, the total length of the midline was compared between groups. The percentage of EdU-positive/DAPI-positive, EdU-positive/KI67-positive, and EdU-positive/KI67-negative MZG from the telencephalic hinge (white dotted outline) is quantified in ( F ), ( G ), and ( H ), respectively. EdU-positive cells within the base of the IHF is quantified in ( I ). EdU-positive/KI67-positive cells or EdU-positive/KI67-negative cells within the IHF were normalised to the total volume of the IHF as quantified in ( J ) and ( K ), respectively. Data represent mean ± SEM, *p<0.05, **p<0.01, ***p<0.001, ns = not significant, as determined with Mann–Whitney tests. ( L ) Schema of major steps involved in IHF remodelling. ( M ) Schema of BTBR phenotype at E15 compared with wildtype C57: BTBR mice display increased proliferation of MZG progenitors and precocious migration to the IHF surface as well as proliferation of the leptomeninges and expansion of the IHF, which may underlie failed IHF remodelling in these mice. See related . Figure 8—source data 1. Measurements of IHF length and cells expressing EdU or KI67 within the telencephalic midline of E13-E15 BTBR and C57 mice.

Article Snippet: Antibody , Mouse monoclonal anti-Glast (EAAT1) , Abcam , Ab49643, RRID: AB_869830 , ‘(1:500)’.

Techniques: Injection, Immunohistochemistry, Marker, MANN-WHITNEY, Migration, Expressing

Journal: eLife

Article Title: DRAXIN regulates interhemispheric fissure remodelling to influence the extent of corpus callosum formation

doi: 10.7554/eLife.61618

Figure Lengend Snippet:

Article Snippet: Antibody , Mouse monoclonal anti-Glast (EAAT1) , Abcam , Ab49643, RRID: AB_869830 , ‘(1:500)’.

Techniques: Recombinant, Sequencing, Mutagenesis, Imaging, Software