Golgi preparations

Golgi preparations

Paulina Carriba and Alun M Davies

The following protocol is based on the protocol provided by FD Rapid GolgiStain Kit (FD NeuroTechnologies).

1. 24 hr before dissection of the brains, prepare the impregnation solution by mixing equal volumes of solution A and B (50:50) in a non-metallic container with lid, and keep it unstirred protected from the light. Prepare enough impregnation solution to cover all brains separately and replace this solution once.
2. Wash 2x PBS the brains dissected from non-perfused mice. Wash 1x milliQ water before to put each brain in a container with lid with enough volume of impregnation solution to cover the brain. Keep the container protected from the light at room temperature.
3. Next day, carefully replace the impregnation solution by fresh impregnation solution without disturbing the brain. Avoid using the bottom impregnation solution in where some precipitates are formed.
4. Keep the brains in impregnation solution for 2 weeks protected from the light at room temperature. Every 4-5 days gently stir without disturbing the brain to allow homogeneous tissue impregnation.
5. Transfer the brain to a new container with lid with enough solution C to cover the brain. Keep it protected from the light at room temperature.
6. Next day, carefully replace the solution C by fresh solution C. Maintain between 3 to 7 days in solution C protected from the light at room temperature.
7. Snap freeze the brain using precooled isopentane to their complete freezing. Brains can be stored at -80°C.
8. Section the brain in a cryostat. Depending on the area to study determine the orientation of the cuts and the thick of the sections. As a reference, for mice hippocampus 150 μm coronal sections, and for mice striatum 100 μm parasagittal sections. Ensure the cryostat temperature is enough down to avoid any thaw. Tissue can be mounted on the specimen disc with optimal cutting temperature (OCT) compound, but not embeded. Do not use the anti-roll plate.

9. Put some drops of solution C on a gelatin-coated microscope slide, and carefully collect the sections with a paintbrush and display them onto the drops of solution C on the gelatin-coated microscope slide.

10. Remove all excess of solution C from the slides with an absorbent tissue. Protected from the light, let it dry at room temperature for at least 24 h to ensure it is completely dry. If it is not completely dry sections are not sufficiently attached to the slide and then could fall down during the process of staining.
11. Staining:

• Wash 2x H₂O, 4 min each.
• Staining 10 min with Solutions D/E [equal parts of Solution D and E and two parts of H₂0 (25 solution D:25 solution E:50 H₂O)].
• Wash 2x H₂O, 4 min each.

• Dehydrate with Ethanol:

  - 50% Ethanol / 50% H₂0: 4 min

  - 75% Ethanol / 25% H₂0: 4 min

  - 95% Ethanol / 5% H₂0: 4 min

  - 4x 100% Ethanol: 4 min each.

• Clear with xylene. 3x xylene 4 min each.

12. Mount sections with Eukitt® Quick-hardening mounting medium or Permount®.

13. Image in bright field microscopy.

14. Slices can be stored at room temperature protected from the light after sealed with polish nail.

15. For analysis of the images, first trace reconstructions using the plugin Simple Neurite Tracer of the Fiji software (Longair et al., 2011). Sholl analysis can be carried out on these reconstructions using the plugin Sholl Analysis of the Fiji software (Schindelin et al., 2012).

Bidimensional tracer reconstruction of striatal neuron from P10 (10 days postnatal) mouse.