Fd rapid golgistain

Only Ctrl and Sev_def animals were used for the Golgi-staining and 3D reconstruction of neurons. To Golgi-stain the right hemispheres, a commercial kit was purchased (FD Rapid GolgiStain™, FD NeuroTechnologies, Inc., Colombia, MD, USA) and the staining performed as per manufacturer’s instructions [52 (link)]. In summary: The hemispheres were stored in A + B solution for 14 days with a solution change after 24 h and then transferred to C solution for seven days with a solution change after 24 h. The hemispheres were then snap-frozen in isopropanol and stored at −80 °C until further processing.
The hemispheres were mounted on a HM 569 M Cryostat (Mikrom, Walldorf, Germany) and the Hip was cut into 200 μm coronal sections in its entirety. The sections were mounted on 2% gelatin-coated slides and left to dry for one hour at RT after which they were rinsed in miliQ water and placed in D + E solution for 10 min, before being dehydrated in alcohol, cleared in xylene and cover slipped.

TRIzol (CAT #15596-018), Nuclease-Free Water (CAT #750023) and Power SYBR® Green PCR Master Mix (CAT # #4367659) were purchased from Thermo Fisher Scientific, Waltham, MA, USA. Vecta stain Elite ABC Kit (Mouse IgG and Rabbit IgG) and Vector SG Peroxidase Substrate Kit were purchased from Vector Laboratories (Burlingame, CA, USA). Entellan® (CAT #107960) was from Merck, Kenilworth, NJ, USA. Mouse anti-neuronal nuclei (NeuN) (Cat# MAB377, RRID: AB_2298772) and rat Horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (CAT #170-6515) were from Bio-Rad Laboratories, Hercules, CA, USA. FD Rapid GolgiStain™ (CAT #PK401) was purchased from FD NeuroTechnologies, Columbia, MD, USA. Rabbit anti-p-CREB and anti-CREB polyclonal antibody (CAT #sc-52, RRID:AB_2629503) were obtained from Cell Signaling Technology, Danvers, Massachusetts, USA. Rabbit anti-vinculin (CAT #129002) were obtained from Abcam, Cambridge, United Kingdom). ECL Western blotting detection reagents (CAT # #RPN2232) were purchased from GE Healthcare, Chicago, IL, USA. 2-chloro-4-((2,5-dimethyl-1-(4-(trifluoromethoxy) phenyl)-1H-imidazol-yl) ethynyl) pyridine (CTEP) was purchased from Axon Medchem. Rabbit anti-mGluR5 (CAT #AB5675) and all other biochemical reagents were purchased from Sigma–Aldrich, Saint Louis, MO, USA.