Tissue tek oct compound medium

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).

The mice were perfused transcardially with saline followed by transfusion with ice-cold 4% neutral buffer paraformaldehyde. The brains were fixed in 4% paraformaldehyde for 72 h, followed by incubation in 20% sucrose for 48 h. After that, the brains were fixed in the frozen O.C.T. compound (Tissue-Tek O.C.T. compound medium, Sakura Finetek United States, Inc., Torrance, CA, United States). The 14 μm coronal plane sections were cut using a CM 3050 S Cryostat (Leica, Berlin Germany). The sections were taken on the gelatin-coated slide and used for further experiments.

For immunofluorescence and histological analysis, as described previously in Reference [40 (link),41 (link)], the mice were transcardially perfused with saline, followed by fixation with 4% ice-cold paraformaldehyde, and the brains were fixed for 72 h, in paraformaldehyde, and then transferred to 20% sucrose, for 72 h. The O.C.T compound (tissue-Tek O.C.T compound medium, Sakura Finetek USA, Inc., Torrance, CA, USA) was used for freezing and blocking the brain, and 14 μm coronal sections were cut, using a CM 3050C cryostat (Leica, Nussloch, Germany). The brain section was taken on a probe-on plus charged slides (Fisher, Rock-ford, IL, USA).

Wire-induced injury of the femoral artery was performed at day 7 of empagliflozin treatment as previously described (19 (link)). In brief, mice were anesthetized as described earlier. A straight spring wire (0.38 mm in diameter, Cook Medical, Bloomington, IN, United States) was then inserted through the profunda femoris artery up to 1 cm into the femoral artery and left in place for 1 min to achieve an adequate wire-induced vessel injury. After removal of the wire, the profunda femoris artery was ligated (7-0 Prolene, Ethicon, Johnson & Johnson, Norderstedt, Germany) and perfusion of the dilated femoral artery was re-established. Mice were sacrificed at 10 or 21 days by cervical dislocation. The femoral artery was carefully harvested and embedded in Tissue-Tek OCT compound medium (Sakura Finetek Europe B.V., Staufen im Breisgau, Germany). Then, the arteries were snap-frozen and stored at –80°C until sectioning.

The animals were euthanized after 12 h of drug treatment to conduct morphological studies as we reported earlier [26 (link)]. Briefly, the brain tissues from all the treated groups after 12 h were subjected to transcardial perfusion with 4 % ice-cold paraformaldehyde. After postfixing these brain tissues, 4 % paraformaldehyde was transferred to 20 % sucrose. The tissues were frozen in OCT (Tissue-Tek O.C.T. Compound Medium, Sakura Finetek USA, Inc., Torrance, CA, USA), sectioned into 14–16-μm sections in the coronal plane with a CM 3050S cryostat (Leica, Wetzlar, Germany). The sections were thaw-mounted on Probe-On positively charged slides (Thermo Fisher Scientific Inc., Waltham, MA, USA).

After cognition assessments, the mice were anesthetized with Rompun (M&S Korea) mixed with Zoletil (M&S Korea) (1:2) (administered; 0.5 mL per 100 g of body weight). For biochemical analysis, the brains were immediately removed, and the hippocampi and cortices regions were dissected out carefully and flash-frozen with liquid nitrogen. For protein extraction, the tissues were mechanically homogenized in PRO-PEP extraction solution on the surface of the ice and centrifuged at 13,000 rpm for 30 min at 4 °C. The supernatants were collected and analyzed by SDS-PAGE. For immunostaining and morphological analysis, the animals were perfused transcardially with normal saline and then, subsequently, with 4% paraformaldehyde (PFA). The brains were removed cautiously, submerged, and fixed at 4 °C in ice-cold PFA for 72 h and were then rinsed with filter-Phosphate-buffered saline (f-PBS) and placed in 20% sucrose solution for 48 h before freezing in OCT compound (Tissue-Tek O.C.T compound medium, Sakura Finetek USA, Inc., Torrance, CA, USA #4583). Serial 14 μm coronal tissue sections were cut on CM 3050C cryostat (Leica, Nussloch, Germany) microtome and thaw-mounted on gelatin-coated slides.