Eclipse 80

The study was performed in accordance with the provisions of the Declaration of Helsinki 1995 (as revised in Edinburgh 2000). We examined paraffin-embedded histological sections from 22 fetuses near term (gestational age approximately 30–38 weeks; crown-rump length [CRL] 250–315 mm). All fetuses were part of the large collection kept at the Department of Anatomy, Universidad Complutense, Madrid, and were the products of miscarriages and ectopic pregnancies at the Department of Obstetrics of the University. All sections were stained with hematoxylin and eosin (H&E). The sectional planes were frontal (3 fetuses), sagittal (12) and horizontal (7). The study was approved by Complutense University ethics committee (B08/374). Observations and photography were usually performed with a Nikon Eclipse 80.

The concentrations of K⁺ ions in the cell suspensions were measured using a K⁺ ion selective electrode, as previously described by Cox et al (30 (link)). The concentration of total free K⁺ in the S. pyogenes suspension was measured following incubation in lysostaphin (50 mg/ml) at 37°C for 30 min, followed by sonication with a Laboratory Continuous Cell Flow Probe Sonicator (SJIA-1500W; Ningbo Yinzhou Sjialab Equipment Co., Ltd., Ningbo, China). Complete lysis was confirmed by microscopic examination (Nikon Eclipse 80).

To assess the number, vitality, and morphology of the sperm, the left epididymis was quickly separated to prepare a sperm suspension. Then, 4 drops of the sperm suspensions were pipetted onto the glass slide and the proportion of motile sperm was observed under a microscope (Nikon eclipse 80; Tokyo, Japan). We observed 10 visual fields for each animal and the average number of dead sperm (500 sperm per field) and the number of dead sperm were also calculated.
The number of sperm was counted under a microscope. A 0.5 mL sperm suspension was taken and diluted with 9.5 mL of saline, and then a 10 µL sperm suspension was transferred into a hemocytometer and stood for 5 minutes.
A drop of the sperm suspension was taken and fixed with methanol for 5 minutes and stained with 1% eosin for 1 hour. Then, the sperm morphology was observed under the microscope after washing and the types of shape of sperm were counted. A total of 1,000 sperm per animal were counted and the deformity rate (%) was calculated. All studies were performed in triplicate.

The study was performed in accordance with the provisions of the Declaration of Helsinki 1995 (as revised in 2013). The study included 45 midterm fetuses of CRL 32–110 mm (GA 8–15 weeks) and 17 late-stage fetuses of CRL 250–328 mm (GA 30–38 weeks). All specimens were part of the large collection kept at the Embryology Institute of the Universidad Complutense, Madrid, and originated from miscarriages and ectopic pregnancies at the Department of Obstetrics of the University. The midterm specimens had been sectioned serially, 10 sagittally and 35 horizontally; most sections were stained with hematoxylin and eosin (H&E), with others stained with Masson trichrome, azan, orange G or silver stain. In contrast, after macroscopic observations during dissection, the abdomens of the late-stage fetuses were sectioned horizontally at 100–200-µm intervals and stained with H&E. During dissection of the late-stage fetuses, photographs were taken with a Pentax K-1 camera equipped with a 50–100 mm zoom lens. Histological samples were observed and most photographs taken with a Nikon Eclipse 80, whereas photographs at ultra-low magnification (objective lens less than ×1) were taken with a high-grade flat scanner and translucent illumination (Epson scanner GTX970, Java, Indonesia). The study protocol was approved by the Ethics Committee of the Universidad Complutense (B08/374).