Journal: International Journal of Molecular Sciences

Article Title: Neurons Induce Tiled Astrocytes with Branches That Avoid Each Other

doi: 10.3390/ijms23084161

Figure Lengend Snippet: Morphology of astrocytes and neurons in co-culture. ( a ) Astrocytes and neurons in rat cerebrum neuron–astrocyte co-culture on DIV15, immunostained by neuronal or astrocytic marker antibodies. Left, astrocytic markers of GLT1 (green), GFAP (red), and GLAST (blue). Middle, a neuronal dendritic marker of MAP2 (green) and astrocytic markers of GAT3 (red) and AQP4 (blue). Right, GLT1 (green), GFAP (red), and phospho-ezrin (blue). Top, scale bar, 100 µm. Bottom, an enlarged view of the area indicated with a square in the left image. Scale bar, 25 µm. Similar results were obtained for 3–7 independently prepared cultures. Images of each channel are provided in . ( b ) High-magnification images of astrocytes and neurons in rat cerebrum neuron–astrocyte co-culture on DIV19. Astrocytic marker GLT1 (green), MAP2 (red), and phospho-ezrin (blue). Scale bar, 10 µm. Similar results were obtained for 3 independently prepared cultures.

Article Snippet: Primary antibodies used for immunostaining were Guinea pig polyclonal anti-GLT1 (GLT1-GP-Af810, Frontier Institute, Hokkaido, Japan, 1:2000 dilution), rabbit polyclonal anti-GLAST (GLAST-Rb-Af660, Frontier Institute, 1:2000 dilution), mouse monoclonal anti-glial fibrillary acidic protein (GFAP) GA5 (GTX73615, Genetex, Irvine, CA, USA, 1:2000 dilution), Guinea pig polyclonal anti-GABA transporter 3 (GAT3) (274 304, Synaptic Systems, Göttingen, Germany, 1:1000 dilution), rabbit polyclonal anti-aquaporin 4 (AQP4) (sc-20812, Santa-Cruz Biotechnologies, Dallas, TX, USA, 1:200 dilution), rabbit monoclonal phospho-Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558) (48G2) (#3726, Cell Signaling Technology, Danvers, MA, USA), chicken polyclonal anti- microtubule-associated protein MAP2 (RRID:AB_2138153, Abcam, Cambridge, UK, 1:5000 dilution), rabbit polyclonal anti-GluA2/3 (RRID:AB_90710, Merck Millipore, Burlington, VT, USA, 1:1000 dilution), mouse monoclonal anti-vGluT1 (RRID: AB_2750766, UC Davis/NIH NeuroMab Facility, Davis, CA, USA, 1:1000 dilution), anti-AQP4 antibody E5415A [ ] (1:10 dilution of culture supernatant, RCB4883, RIKEN BRC, Ibaraki, Japan).

Techniques: Co-Culture Assay, Marker

Journal: International Journal of Molecular Sciences

Article Title: Neurons Induce Tiled Astrocytes with Branches That Avoid Each Other

doi: 10.3390/ijms23084161

Figure Lengend Snippet: Time-course of astrocytic and neuronal morphological and functional maturation. ( a ) The time-course of neuronal dendrite growth and astrocytic branching and protein expression. Top, MAP2 (red), GLAST (green), GFAP (blue); middle, GluA2/3 (green), vGluT1(magenta); bottom, GLAST (red), GAT3(green), GLT1 (blue). Scale bar, 200 µm. Similar results were obtained for 3 independently prepared cultures. Images of each channel are provided in . ( b ) The time-course of neuronal or astrocytic marker positive area. The sizes of each neuronal or astrocytic marker area, with fluorescence signal intensity above the threshold. The values of each marker protein are normalized to that on DIV5. Similar results were obtained for 3 independently prepared cultures. ( c ) The time-course of astrocytic marker expression calculated as the average signal intensity of each marker protein on astrocytes. Each astrocytic marker’s average fluorescence signal intensity above the threshold was calculated. The average fluorescence intensities of each astrocytic marker were normalized to that on DIV5. Similar results were obtained for 3 independently prepared cultures. See a for AQP4 images.

Article Snippet: Primary antibodies used for immunostaining were Guinea pig polyclonal anti-GLT1 (GLT1-GP-Af810, Frontier Institute, Hokkaido, Japan, 1:2000 dilution), rabbit polyclonal anti-GLAST (GLAST-Rb-Af660, Frontier Institute, 1:2000 dilution), mouse monoclonal anti-glial fibrillary acidic protein (GFAP) GA5 (GTX73615, Genetex, Irvine, CA, USA, 1:2000 dilution), Guinea pig polyclonal anti-GABA transporter 3 (GAT3) (274 304, Synaptic Systems, Göttingen, Germany, 1:1000 dilution), rabbit polyclonal anti-aquaporin 4 (AQP4) (sc-20812, Santa-Cruz Biotechnologies, Dallas, TX, USA, 1:200 dilution), rabbit monoclonal phospho-Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558) (48G2) (#3726, Cell Signaling Technology, Danvers, MA, USA), chicken polyclonal anti- microtubule-associated protein MAP2 (RRID:AB_2138153, Abcam, Cambridge, UK, 1:5000 dilution), rabbit polyclonal anti-GluA2/3 (RRID:AB_90710, Merck Millipore, Burlington, VT, USA, 1:1000 dilution), mouse monoclonal anti-vGluT1 (RRID: AB_2750766, UC Davis/NIH NeuroMab Facility, Davis, CA, USA, 1:1000 dilution), anti-AQP4 antibody E5415A [ ] (1:10 dilution of culture supernatant, RCB4883, RIKEN BRC, Ibaraki, Japan).

Techniques: Functional Assay, Expressing, Marker, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Neurons Induce Tiled Astrocytes with Branches That Avoid Each Other

doi: 10.3390/ijms23084161

Figure Lengend Snippet: The correlation between a pair of transporter protein expression levels for each astrocyte. Immunofluorescence signal intensities of each transporter protein for each astrocyte were measured as average signal intensity on the astrocyte cell body. The correlation coefficients between transporter protein signal intensities for all astrocytes in each image were calculated. Average correlation coefficients of independently prepared samples ( n ), with 65–126 cells for each analysis.

Article Snippet: Primary antibodies used for immunostaining were Guinea pig polyclonal anti-GLT1 (GLT1-GP-Af810, Frontier Institute, Hokkaido, Japan, 1:2000 dilution), rabbit polyclonal anti-GLAST (GLAST-Rb-Af660, Frontier Institute, 1:2000 dilution), mouse monoclonal anti-glial fibrillary acidic protein (GFAP) GA5 (GTX73615, Genetex, Irvine, CA, USA, 1:2000 dilution), Guinea pig polyclonal anti-GABA transporter 3 (GAT3) (274 304, Synaptic Systems, Göttingen, Germany, 1:1000 dilution), rabbit polyclonal anti-aquaporin 4 (AQP4) (sc-20812, Santa-Cruz Biotechnologies, Dallas, TX, USA, 1:200 dilution), rabbit monoclonal phospho-Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558) (48G2) (#3726, Cell Signaling Technology, Danvers, MA, USA), chicken polyclonal anti- microtubule-associated protein MAP2 (RRID:AB_2138153, Abcam, Cambridge, UK, 1:5000 dilution), rabbit polyclonal anti-GluA2/3 (RRID:AB_90710, Merck Millipore, Burlington, VT, USA, 1:1000 dilution), mouse monoclonal anti-vGluT1 (RRID: AB_2750766, UC Davis/NIH NeuroMab Facility, Davis, CA, USA, 1:1000 dilution), anti-AQP4 antibody E5415A [ ] (1:10 dilution of culture supernatant, RCB4883, RIKEN BRC, Ibaraki, Japan).

Techniques: Expressing, Immunofluorescence