Journal: iScience

Article Title: Astrocytic FABP5 mediates retrograde endocannabinoid transport at central synapses

doi: 10.1016/j.isci.2025.112342

Figure Lengend Snippet:

Article Snippet: The following AAVs were used: AAV-CAG-FABP5 expressing mouse FABP5 under the CAG promoter (Duke VVC #pBK366, serotype AAV9), AAV-CAG-FABP5 MUT expressing FABP5 MUT (Duke VVC #1937, serotype AAV9), AAV-CAG-FABP5 SEC expressing FABP5 SEC (Duke VVC #pBK383, serotype AAV9), AAV-CAG-FABP7 expressing mouse FABP7 (Duke VVC #pBK1433, serotype AAV9), AAV-CMV-Cre (Addgene, #105545, serotype AAV9), AAV-CMV-GFP (Addgene, #105530, serotype AAV9), AAV-hSyn-Cre (Addgene, #105540, serotype AAV5), pAAV-hSyn-EGFP (Addgene, #50465, serotype AAV5), AAV-gfaABC1D-FABP5 expressing FABP5 under the astrocyte-specific gfaABC1D promoter (Duke VVC #pBK1762, serotype AAV8), pAAV.GFAP.eGFP.WPRE.hGH (Addgene, #105549, serotype AAV5), and AAV-hSyn-FABP5 (Duke VVC #pBK1761, serotype AAV5).

Techniques: Virus, Recombinant, Software, Imaging, Transfection, Real-time Polymerase Chain Reaction, SYBR Green Assay, Mass Spectrometry, Control, Microscopy

Journal: bioRxiv

Article Title: Improved transfection methods of primary cultured astrocytes for observation of cytoskeletal structures

doi: 10.1101/2025.01.27.635031

Figure Lengend Snippet: (A) Lower magnification view of astrocytes cultured at high density (initial cell density of 0.6 × 10 5 cells/cm 2 ; cultured for 7 days) and low density (initial cell density of 0.3 × 10 5 cells/cm 2 ; cultured for 6 days). Phase contrast images show cells just before trypsin treatment (pre-transfection) and cultured for one day after electroporation. Scale bar = 200 μm. (B-D) Representative images of (B) EGFP-ezrin and mCherry-actin, (C) EGFP-actin and mCherry-lasp-2, and (D) EGFP-ezrin and mCherry-lasp-2 at 2, 3, and 2 days post-transfection, respectively. See supplementary movie 1 for (D); (D’) kymographs of magnified segment of stem process surface from supplemental movie 1 with a sequence interval of 20 sec. Arrows in (C) and (D) indicate examples of elliptical structures of fluorescent-tagged lasp-2. Asterisks in (D’) indicate examples of structures where lasp-2 and ezrin colocalized.

Article Snippet: The regions coding EGFP of the pLenti CMV EGFP Puro (658-5) vector (Addgene) were removed using BamHI and SalI and replaced with PCR products using NEBuilder HiFi DNA Assembly (New England BioLabs, MA, USA).

Techniques: Cell Culture, Transfection, Electroporation, Sequencing

Journal: bioRxiv

Article Title: Improved transfection methods of primary cultured astrocytes for observation of cytoskeletal structures

doi: 10.1101/2025.01.27.635031

Figure Lengend Snippet: (A) Lower magnification view of astrocytes before and after (1d, 2d and 5d) infection. Scale bar = 200 μm. (B-E) Representative images of (B) EGFP, (C) EGFP-lasp-2, (D) mCherry-lasp-2, and (E) mCherry-lasp-2 and added Sara Fluor 497 actin probe in astrocytes at 7, 4, 6, and 5 days post-infection with lentivirus vectors, respectively. (F) Time-lapse imaging of astrocytes infected with lentiviral coding with EGFP or EGFP-lasp-2 every 2 hours. Arrows in (C) to (E) indicate examples of elliptical structures of fluorescent-tagged lasp-2.

Article Snippet: The regions coding EGFP of the pLenti CMV EGFP Puro (658-5) vector (Addgene) were removed using BamHI and SalI and replaced with PCR products using NEBuilder HiFi DNA Assembly (New England BioLabs, MA, USA).

Techniques: Infection, Imaging