Perfusion-Based Brain Tissue Preparation

After completion of behavioral studies, For morphological/immunofluorescence analysis, the experimental mice/animals (n = 8) were brought into the surgical room and anesthetized with Rompun (Xylazine; 0.05 mL/100 g body weight) and Zoletil (ketamine; 0.1 mL/100 g body weight) as described previously [6 (link)]. These mice were perfused transcardially with normal saline solution (0.9%) followed by paraformaldehyde (4%). The mice brain tissue was removed immediately and fixed at 4 °C for 72 h with ice-cold paraformaldehyde. After this, they were submerged for 72 h in sucrose phosphate buffer (20%). All mice’s brains were frozen in optimum cutting temperature (O.C.T) compound (tissue-Tek O.C.T compound medium, Sakura Finetek USA, Inc., Torrance, CA, USA) and further cut into coronal sections of 14 μm using a CM3050C cryostat (Leica, Nussloch, Germany).