Permounttm

Golgi staining was performed using the Golgi Staining Kit (FD NeuroTechnologies) according to the manufacturer’s instructions. All procedures were performed under dark conditions. Brains hemispheres used for Golgi-cox staining were immersed in 3 mL mixtures of equal parts of kit solutions A and B and stored at room temperature for 2 weeks. Then, brain tissues were stored in solution C at room temperature for at least 48 h and up to 7 days before sectioning. Solutions A, B, and C were renewed within the first 24 h. Coronal sections of 100 mm were cut with a cryostat (Thermo Fisher Scientific, CryoStar NX50). Each section was mounted with solution C on an adhesive microscope slide precoated with 1% gelatin/0.1% chromalaun on both slides and stained according to the manufacturer’s protocol with the exception that AppliClear (AppliChem) was used instead of xylene. Finally, slices were coverslipped with PermountTM (Thermo Fisher Scientific).

We adapted a Golgi-staining protocol using reagents from the superGolgi Kit (Bioenno Tech, Santa Ana, CA, USA) [52 (link)]; Golgi-staining is a reliable method for assessing dendrites and dendritic spine dynamics [30 (link),53 (link)]. Right hemispheres were immediately impregnated in a potassium dichromate solution for 2 weeks (n = 6). Next, sections were immersed for at least 48 h in a post-impregnation buffer. Samples were sectioned at 200 µm in 1× PBS along the coronal plane; they were then transferred into wells and washed with 0.01 M PBS-T buffer (pH 7.4, 0.3% Triton X-100). Immediately after washing, samples were stained with ammonium hydroxide and then immersed in a post-staining buffer. Sections were again washed with PBS-T, mounted on 1% gelatin-coated slides, and allowed to dry. The sections were then dehydrated with ethanol solutions, cleaned with xylene, and cover-slipped with Permount^TM (Thermo Fisher Scientific, Waltham, MA, USA).

In order to look at dendrite morphology and spine dynamics, Golgi staining was used to randomly stain neurons in the hippocampus [44 (link)]. An adapted protocol was used with the reagents of the superGolgi kit (Bioenno Tech LLC, Santa Ana, CA, USA) [45 (link)]. Right hemispheres were impregnated with potassium dichromate solution for two weeks (n = 32) followed by 48 h in post-impregnation buffer. The brain was sectioned in the coronal plane at 200 µm thick slices using a vibratome. The slices were transferred into a 24-well plate for staining. The slices were washed in PBS-T (1× PBS, 0.3% Triton^TM X-100) before staining with ammonium hydroxide and washed in post-stain buffer. After another PBS-T wash, the slices were mounted on 1% gelatin-coated slides and left to dry overnight in Coplin jars. The next morning, the slides were dehydrated with ethanol, cleaned in xylene, and cover-slipped with Permount^TM, Thermo Fisher Scientific^TM Inc., Waltham, MA, USA).

Shortly after behavioral testing, animals were euthanized, and their brains were collected at day 11 post-8.5 Gy and dissected along the midsagittal plane and half of the hippocampus was harvested for Golgi staining. The Golgi method of staining has long proven to be a reliable method for assessing dendrite and dendritic spine dynamics due to various treatments, because of its resistance to fading or photobleaching over time [65 (link),66 (link)]. We adapted a staining protocol and used the reagents contained in the superGolgi kit (Bioenno Tech, Santa Ana, CA, USA) [67 (link)]. Right hemispheres were immediately impregnated in a potassium dichromate solution for two weeks (n = 5). Next, sections were immersed for at least 48 h in a post-impregnation buffer. Samples were sectioned at 200 µm in 1× PBS along the coronal plane. Samples were then transferred into wells and washed with 0.01 M PBS buffer (pH 7.4) with Triton X-100 (0.3%) (PBS-T). Immediately after washing, samples were stained with ammonium hydroxide and then immersed in a post-staining buffer. Sections were again washed in PBS-T, mounted on 1% gelatin-coated slides, and allowed to dry. Sections were finally dehydrated with ethanol solutions, followed by cleaning in xylene, and coverslipped with Permount^TM (Thermo Fisher Scientific, Waltham, MA, USA).