Hm525

Following the habituation phase, all animals were deeply anesthetized with an intraperitoneal injection of pentobarbital (Morbital, Biowet, Poland; 2 mL/kg body weight), and after cessation of breathing, immediately perfused transcardially with saline (0.9%) followed by 4% paraformaldehyde (pH 7.4; 1040051000, Sigma Aldrich, Taufkirchen, Germany) in phosphate-buffered saline (PBS; P5493, Sigma Aldrich, Taufkirchen, Germany). After perfusion, brains were carefully dissected from the skulls and post-fixed by immersion in the same fixative for 24 h, washed twice in 0.1 M phosphate buffer (pH = 7.4, 4 °C), and then stored for 3–5 days in graded solutions (19% and 30%) of sucrose (S0389, Sigma Aldrich, Saint Louis, MO, USA) in 1×PBS at 4 °C until full saturation. Finally, the brains were frozen and then coronally sectioned at a thickness of 10 μm using a cryostat (HM525 Zeiss, Germany). The sections were mounted on object slides and stored at −80 °C until further processing.

Following the habituation phase, all animals were deeply anesthetized with an intraperitoneal injection of pentobarbital (Morbital, Biowet, Poland; 2 mL/kg body weight), and after cessation of breathing, immediately perfused transcardially with saline (0.9%) followed by 4% paraformaldehyde (pH 7.4; 1040051000, Sigma Aldrich, Taufkirchen, Germany) in phosphate-buffered saline (PBS; P5493, Sigma Aldrich, Taufkirchen, Germany). After perfusion, brains were carefully dissected from the skulls and post-fixed by immersion in the same fixative for 24 h, washed twice in 0.1 M phosphate buffer (pH = 7.4, 4 °C), and then stored for 3–5 days in graded solutions (19% and 30%) of sucrose (S0389, Sigma Aldrich, Saint Louis, MO, USA) in 1×PBS at 4 °C until full saturation. Finally, the brains were frozen and then coronally sectioned at a thickness of 10 μm using a cryostat (HM525 Zeiss, Germany). The sections were mounted on object slides and stored at −80 °C until further processing.

After the habituation phase, all SHRs and WKYs were randomly allocated into seven age groups in accordance with the study plan, with all animals from each group later given the same tissue processing. Briefly, the rats were acutely anesthetized with an intraperitoneal inoculation of Morbital (Biowet, Puławy, Poland; 2 ml/kg, 133.3 mg/ml of pentobarbital sodium salt and 26.7 mg/ml of pentobarbital). Following cessation of breathing, animals were immediately perfused transcardially with saline (0.9%) followed by 4% paraformaldehyde (PFA; pH 7.4 (1040051000; Merck, Darmstadt, Germany), which was dissolved in phosphate-buffered saline (PBS) (P5493; Sigma-Aldrich, Schnelldorf, Germany). After perfusion, the whole brain was conscientiously removed from the skull. In the next step, brains were post-fixed by immersion in 4% PFA for 24 h and then rinsed three times in 0.1 M phosphate buffer (pH 7.4, 4°C). Thereafter, all brains were cryoprotected in series (10%, 20%, and 30%) with sucrose (363-117720907; Alchem, Wrocław, Poland) in 1× PBS at 4°C until they sunk (3–5 days). Conclusively, the brains were frozen as blocks and were then coronally cut at a thickness of 10 μm using a cryostat (HM525; Zeiss, Jena, Germany). The tissue sections were placed on glass slides and stored at −80°C until further investigation.