Jan 16, 2026
Golgi-Cox stain and neuronal morphological analysis
- Mario Alberto Bautista-Carro1
- 1Cinvestav
External link: https://doi.org/10.1371/journal.pone.0339028
Protocol Citation: Mario Alberto Bautista-Carro 2026. Golgi-Cox stain and neuronal morphological analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7n643lwz/v1
Manuscript citation:
Bautista-Carro MA, Sánchez-Teoyotl P, Juárez-Serrano D, Iannitti T, Díaz A, Flores G, Morales-Medina JC (2026) Olfactory bulbectomy induces neurobiological alterations in the prefrontal cortex and hyperlocomotion in male rats. PLOS One 21(1). doi: 10.1371/journal.pone.0339028
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 09, 2025
Last Modified: January 16, 2026
Protocol Integer ID: 229355
Keywords: Golgi-Cox method, Neuronal morphology, Neuron reconstruction, Dendritic spines, Morphometric analysis, Sholl analysis, neuronal morphological analysis the golgi, cox stain on rat brain, detailed study of neuronal morphology, neuronal morphological analysis, neuronal morphology, neuronal organization, golgi, cox stain, performing golgi, help of optical microscopy, optical microscopy, rat brain, dendritic arborization, spine density
Abstract
The Golgi-Cox technique allows for detailed study of neuronal morphology. With the help of optical microscopy and Sholl analysis, dendritic arborization, spine density, and neuronal organization can be analyzed. This allows us to identify structural changes in physiological or pathological conditions. Below we describe protocol for performing Golgi-Cox stain on rat brains and Sholl analysis.
Guidelines
The entire tissue preparation process must be carried out in a darkroom. Be sure to work in a laboratory fume hood when necessary.
Materials
Perfusion pump, Ketamine, Xylazine, Sterile saline solution, 0.9% saline solution, Syringes, Falcon tubes, Golgi-Cox solution, Vibratome, Gelatinized slides, Ammonium hydroxide, Kodak film fixer, Alcohol, Xylene, Light microscope equipped with camera lucida, Permount mounting medium, Cover slips.
Troubleshooting
Before start
Ensure you have all the necessary laboratory materials and equipment.
Perfusion
Anesthetize the rats by intraperitoneal injection of a ketamine/xylazine cocktail (0.75 ml ketamine + 0.25 ml xylazine + 5 ml sterile saline), administering a dose of 0.125 ml per 20 g of body weight.
Perfuse the rats intracardially with 0.9% saline solution and quickly remove the brain.
Tissue preparation
4w 5d
Place the brains in Golgi-Cox solution for 30 days.
4w 2d
Incubate 30% sucrose solution for 3 days.
3d
Using a vibratome (Leica, VT1000S microsystem, USA), obtain 200 µm coronal sections of the brain region of interest and mount them on 2% gelatinized slides. Keep the slides in a humid chamber.
Staining procedure (in darkroom)
1h 45m 25s
Rinse with distilled water 5 times.
5s
Treat sections with ammonium hydroxide for 30 minutes.
30m
Rinse with distilled water 10 times.
10s
Incubate for 30 minutes in Kodak Film Fixer.
30m
Rinse with distilled water 10 times and dehydrate in increasing concentrations of alcohol: 75% 5 minutes, 90% 5 minutes, and 100% 10 minutes twice.
30m 10s
Incubate with xylene for 15 minutes.
15m
Cover the slides with Permount mounting medium and coverslips.
Neuronal morphological analysis
Using an optical microscope (DM 2000 microscope, Leica Microsystems, USA) equipped with a camera lucida, reconstruct ten pyramidal neurons (5 in each hemisphere) from each brain in a two-dimensional plane.
Quantify dendritic tracing by Sholl analysis. Place a transparent grid with concentric rings spaced 10 µm apart over the dendritic drawing (centering the soma) and count the number of intersections. Calculate the total dendritic length by multiplying the total number of intersections in each ring by 10 µm. Calculate the dendritic arborization as the total number of dendritic branches present in each order of the dendritic axis.
To obtain the density of spines, count and draw the spines of ten distal and/or last-order dendrites with an approximate length of 10 µm (5 for each hemisphere) at 1000x magnification.
Protocol references
Flores, G., Morales-Medina, J. C., Rodríguez-Sosa, L., 26 Calderón-Rosete, G. (2015). Characterization of Cytoarchitecture of dendrites and fiber neurons using the golgi-cox method: An overview. Horizons in neuroscience research, 137-146.
Espinoza, I., Gómez-Villalobos, M. J., Aguilar-Hernández, L., Flores, G., 26 Morales-Medina, J. C. (2025). Cerebrolysin treatment improved short-term memory deficits while simultaneously increasing hippocampal spine density in hypertensive female rats. Behavioural brain research, 481, 115436.
Sholl D. A. (1953). Dendritic organization in the neurons of the visual and motor cortices of the cat. Journal of anatomy, 87(4), 387–406.