Microtome

Twenty-four hours after the last drug administration, animals were anaesthetized and perfused with 4% paraformaldehyde PBS (pH 7.2–7.4). The hippocampi with an ipsilateral retrosplenial cortex were quickly excised, postfixed, dehydrated, embedded in paraffin, and finally sectioned on the microtome (Leica Microsystems, Wetzlar, Germany) in the coronal plane (−2.00 to −2.80 mm from bregma) at 7 μm thick slices, according to Paxinos & Watson’s The Rat Brain in Stereotaxic Coordinates (2007) [71 ].

The fixed jejunum-tissue samples were dehydrated in a tissue processor (Leica Microsystems K. K., Tokyo, Japan) and embedded in paraffin wax as previously described^{38 (link)}. The embedded tissue was cut into 5-μm sections using a microtome (Leica Microsystems K. K.) and mounted on polylysine-coated glass slides (Boster Corporation, China). Hematoxylin-Eosin (HE) staining was performed using a routine protocol for histological and histopathological analyses. Histopathological examinations were conducted by light microscopy and findings were imaged and analyzed using a pathological image analysis system (Leica Qwin, Jiangsu, China) with a digital camera (DP72; Olympus). The pathological grade of the jejunum was evaluated by summing the evaluated scores of three factors:
(i) Inflammation (score 0–3): 0 = no inflammatory-cell infiltration; 1 = slight inflammatory-cell infiltration; 2 = moderate inflammatory-cell infiltration; 3 = severe inflammatory-cell infiltration. (ii) Extent of lesions (score 0–3): 0 = No lesion; 1 = Lesion in the mucosal layer; 2 = Lesion in the mucosal layer and submucosa; 3 = Transparent cell wall. (iii) Crypt Damage (score 0–4): 0 = No lesion in crypt; 1 = 1/3 crypt lesion; 2 = 2/3 crypt lesion; 3 = only the epithelial surface was intact; 4 = Crypt and epithelium not visible.

To eliminate tannin-like substances that may be mistaken for the rubber inclusion in laticifer, bark samples were fixed in 80% ethanol for 24 h at room temperature, treated with iodine and bromine in glacial acetic acid [1 ], and embedded in paraffin after dehydration. Sections (12 μm in thickness) were cut with a microtome (Leica Microsystems Inc., Bannockburn, IL, Germany) and stained with fast green. The laticifers in sections could be recognized because the rubber in the laticifers was brown in color due to the iodine-bromine treatment [1 ].

The fixed brains were cut into 2-cm³ segments, placed in a tissue processor apparatus (Shanon-Elliott, USA) and processed in the following order: 10% formalin for 6 h, 96% ethanol for 3 h, 100% ethanol for 3 h, xylene for 3 h, and paraffin 63°C for 7 h. The processed segments were molded by paraffin 63°C in metal boxes. The paraffin sections were cut into 5 µm-thick slices by a microtome (Leica instruments, Germany) and stained with hematoxyline and eosin[20 (link)]. The cells in CA1, CA3, and dendate gyrus, which had bright and healthy nucleus, were counted manually using a stereoscopic microscope (Olympus, Japan) and a light microscope (Zeiss, Germany). Digital images were taken by a video camera linked to a computer with Motic images software (Motic China Group Co., China). The slices were between -3.24 mm and -5.88mm distance from bregma.

Two separate sections of the left lung were used for histopathological analysis. Lung tissues were fixed in 4% paraformaldehyde, embedded in paraffin and sliced into 5-μm-thick sections with a microtome (Leica Microsystems, Ontario, Canada). Lung sections were adhered to a microscope slide and stained with hematoxylin and eosin. Images were acquired by a Panoramic DESK slide scanner (3DHISTECH, Hungary) and analyzed using CaseViewer (3DHISTECH, Hungary). Five random high-power fields (40 × magnification) per mouse lung were analyzed in a blinded fashion for histological lung injury and scored as per the American Thoracic Society guidelines for experimental ALI [39 (link)].