Issue	Potential causes and solutions
Poor or nonexistent virus expression	Missing viral expression may indicate unsuccessful microinjection, most likely due to clogging of microinjector. Another possibility is decreased transduction efficiency of a virus due to poor storage conditions or too many thaw/freezing cycles.
Saturated virus expression	Some anatomically insulated brain structures (like hippocampus) sometimes show Lck-GFP signal saturation. The virus used in this protocol can be diluted with saline if needed to produce optimal expression
Poor antibody penetration	Poor perfusion and/or too long period of post-fixation (2–4h max). Folding of tissue during antibody incubation period. For colocalization antibodies other than those referenced here, the incubation time and/or level of detergent used during IHC can be varied.
Ambiguity regarding the number of collected astrocytes	Sometimes it can be hard to assess if the collected astrocyte is indeed singular using only visual observation. In these instances, colocalization analyses between Lck-GFP and DAPI as well as Lck-GFP and GFAP can be beneficial. A single astrocyte can be colocalized with multiple nuclei but only one of them should be encapsulated in GFAP signal.
Diminishing fluorescence intensity along z-axis	Sometimes acquired Lck-GFP signal shows clear decrease in fluorescence signal intensity along the z-axis due to diminishing excitation potential of a light source deeper in the tissue. Since this can affect surface building, the signal should be attenuated along the z-axis with a built-in Imaris XTension called “Attenuation Correction”. Imaris XTensions are scripts that compliment Imaris analysis, enabling features that are not yet part of a stand-alone Imaris experience. This is achieved by running a part of a code outside Imaris ecosystem (in MATLAB) and importing results back in. Attenuation correction itself is a part of a default Imaris installation package and does not require an additional installation but it does require a working MATLAB installation. See Figure 6 for comparison. The same correction can be applied to colocalization signal or any other IHC staining.