Astrocytes display complex morphology ranging from multiple main processes stemming from the soma to endfeet and a myriad of leaflets that are in close contact with synapses and other cellular structures. This extended feature, which is heterogeneous across brain regions and physiological states, is pivotal to understanding their dynamic interactions with neighboring neurons and glia. Therefore, assessing astrocytic structures is crucial to understanding their roles in brain networks. Immunostaining for Glial Fibrillary Acidic Protein (GFAP), an astrocyte-specific cytoskeletal protein, is commonly used to analyze the astrocytic backbone. However, staining efficiency varies with experimental conditions used during the histological processing. This study investigates parameters affecting the visualization of astrocytic backbone reconstruction in mouse hippocampal slices: slicing method and thickness and antigen retrieval. We assessed astrocytic morphometric features such as process number, length, and arbor complexity of GFAP-labeled backbones. We compared the use of FIJI-SNT and IMARIS software, evaluating the reliability of capturing the astrocyte structure under different conditions. This comprehensive analysis suggests more reliable protocols and conditions for optimal visualization of astrocytic structures.