To analyze astrocytes, optical sections of Lck-GFP-expressing cells were collected using a Zeiss LSM 800 confocal-scanning microscope (405, 488, 561, and 640 nm diode lasers; 2 Gallium Arsenide Phosphide (GaAsP) detectors) with a 63× oil-immersed objective (Zeiss, Oberkochen, Germany) and ZEN software suite (ZEN 2.3 (blue edition), Zeiss). Frame size was set to 1,024 × 1,024 pixels, bit depths to 16-bit, averaging to 4×, and z-step size to 1 μm. The imaging was restricted to previously mentioned regions (as marked in Figures 1 , 2
After acquisition, raw images were transferred to an image-processing workstation and deconvolved using AutoQuant software (v. X3.0.4, MediaCybernetics). A blind deconvolution algorithm with 10 iterations was run on each z-stack. The deconvolved output stack was directly imported to Imaris software (v. 8.4.1, Bitplane, Zürich, Switzerland) which was used to generate a 3-dimensional reconstruction of each individual astrocyte. Each individual cell was then isolated and a surface was built around it using Lck-GFP innate fluorescence signal. Surface area and volume were extracted from each individually built astrocyte surface. A special masked channel was generated using these surfaces, completely isolating the astrocyte Lck-GFP signal from Lck-GFP background. The masked channel was then used to perform colocalization analysis between the astrocyte Lck-GFP signal and the Alexa 594 signal, representing the PSD-95 post-synaptic neuronal marker. To remove the background, threshold for the PSD-95 channel was manually selected by taking repeated measurements of unambiguous puncta of fluorescent signal intensity on multiple optical slices. An average of these measurements was used as a final threshold for the PSD-95 channel. A new colocalization channel was generated which provided percentage of region of interest (ROI) colocalized with the PSD-95 channel (ROI was set as a masked Lck-GFP channel). It should be emphasized that fluorescence colocalization analysis is not a tool for detecting molecular interactions, but rather for assessing co-registry of two or more fluorophores within a given voxel. Accordingly, colocalization of Lck-GFP and PSD-95 signals serves as a proxy of astrocyte proximity to the neuronal post- synaptic terminal. The latter was checked by automatically counting PSD-95 puncta above a predetermined threshold within assigned 25 μm × 25 μm × 25 μm box for a subset of samples. Slides and files were encrypted during acquisition and manipulation to assure unbiased processing.
After acquisition, raw images were transferred to an image-processing workstation and deconvolved using AutoQuant software (v. X3.0.4, MediaCybernetics). A blind deconvolution algorithm with 10 iterations was run on each z-stack. The deconvolved output stack was directly imported to Imaris software (v. 8.4.1, Bitplane, Zürich, Switzerland) which was used to generate a 3-dimensional reconstruction of each individual astrocyte. Each individual cell was then isolated and a surface was built around it using Lck-GFP innate fluorescence signal. Surface area and volume were extracted from each individually built astrocyte surface. A special masked channel was generated using these surfaces, completely isolating the astrocyte Lck-GFP signal from Lck-GFP background. The masked channel was then used to perform colocalization analysis between the astrocyte Lck-GFP signal and the Alexa 594 signal, representing the PSD-95 post-synaptic neuronal marker. To remove the background, threshold for the PSD-95 channel was manually selected by taking repeated measurements of unambiguous puncta of fluorescent signal intensity on multiple optical slices. An average of these measurements was used as a final threshold for the PSD-95 channel. A new colocalization channel was generated which provided percentage of region of interest (ROI) colocalized with the PSD-95 channel (ROI was set as a masked Lck-GFP channel). It should be emphasized that fluorescence colocalization analysis is not a tool for detecting molecular interactions, but rather for assessing co-registry of two or more fluorophores within a given voxel. Accordingly, colocalization of Lck-GFP and PSD-95 signals serves as a proxy of astrocyte proximity to the neuronal post- synaptic terminal. The latter was checked by automatically counting PSD-95 puncta above a predetermined threshold within assigned 25 μm × 25 μm × 25 μm box for a subset of samples. Slides and files were encrypted during acquisition and manipulation to assure unbiased processing.
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