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Semen Analysis
Published in Botros Rizk, Ashok Agarwal, Edmund S. Sabanegh, Male Infertility in Reproductive Medicine, 2019
Meaghanne K. Caraballo, Alyssa M. Giroski, Rakesh Sharma, Ashok Agarwal
The staining solution consists of Eosin–Y (1%) and Nigrosin (10%). The slides are labeled and one drop of liquefied semen sample is placed into a Boerner slide well. Two drops of Eosin are added and mixed well followed by two drops of Nigrosin. After mixing well, two smears are made and air dried and a coverslip is placed and sealed with a cytoseal mounting media. The slide is observed under 100× objective. Eosin stains only the dead sperm, turning them a dark pink, whereas live sperm appear white or faint pink (Figure 3.2). The normal range is 58% according to WHO guidelines [3]. For specimens with motility <25%, viability should be ≥25%.
Evaluation of sperm
Published in David K. Gardner, Ariel Weissman, Colin M. Howles, Zeev Shoham, Textbook of Assisted Reproductive Techniques, 2017
Kaylen M. Silverberg, Tom Turner
An intact plasma membrane is an integral component of, and possibly a biologic/diagnostic indicator for, sperm viability. The underlying principle is that viable sperm contain intact plasma membranes that prevent the passage of certain stains, whereas nonviable sperm have defects within their membranes that allow for staining of the sperm. Several so-called “vital stains” have been employed for this purpose. They include eosin Y, trypan blue, and/ or nigrosin (27). When viewed with either bright field or phase contrast microscopy, these stains allow for the differentiation of viable, non-motile sperm from dead sperm. This procedure may, therefore, play a significant role in determining the percentage of immotile sperm that are viable and available for ICSI. Unfortunately, dyes such as eosin Y are specific DNA probes that may have toxic effects if they enter a viable sperm or oocyte, which precludes the use of these sperm that have been exposed to the dyes for ICSI or insemination. Flow cytometry has also been utilized for the determination of sperm viability. Like vital staining, flow cytometry is based on the principle that an intact plasma membrane will prevent the passage of nucleic acid-specific stains. Some techniques, such as the one described by Noiles et al., employ dual staining, which can differentiate between an intact membrane and a damaged membrane (28). There are no studies that prospectively evaluate sperm viability staining as a predictor of ART outcome.
Tissue Staining Techniques for Stroke Studies
Published in Yanlin Wang-Fischer, Manual of Stroke Models in Rats, 2008
Yanlin Wang-Fischer, Lee Koetzner
Note: Brain sections must be defatted (see step 3) before H&E staining. The 4% PFA-perfused brains are sliced in a cryostat microtome at 40-μm thickness.The slices are mounted on subbed glass slides in order and air dried overnight.Defatting the brain sections: Sections are soaked for at least 1 hour in a mixture of chloroform and 100% ethanol (1:1).Sections are treated by successive 5-minute washes in 100%, 95%, 70%, and 50% EtOH and are rinsed under tap water (the successive washes are to rehydrate the sections). Note: Wait until the sections dry a little bit (no water is dripping). If the slides had been prepared several days before, the slides must be put into double-distilled water for 5 minutes before staining.Staining procedure: Acid hematoxylin solution (eosin-hematoxylin solution, Sigma-Aldrich, Cat. No. 2852) is applied over the tissue slides and stained for 20 to 25 minutes. The process is monitored visually, by eye or under a microscope, and is sufficient when the cell nuclei turn blue.Wash with tap water twice.The slides are washed gently (to prevent tissue dislodgement) in 0.25% ammonia solution for about 10 seconds.Wash with tap water twice.Eosin Y solution is applied over the tissue slides (solution alcoholic, Sigma-Aldrich, Cat. No. HT110116) for 10 seconds.Wash with tap water twice.Dehydrate with 95% alcohol, EtOH, twice for 5 minutes each.Continue to dehydrate with 100% EtOH for 5 minutes.The tissue slides are placed into xylene or xylene substitute solution for 5 minutes (Thermo Electron, Cat. No. 9990505).
Metabolomics reveals the effects of hydroxysafflor yellow A on neurogenesis and axon regeneration after experimental traumatic brain injury
Published in Pharmaceutical Biology, 2023
En Hu, Teng Li, Zhilin Li, Hong Su, Qiuju Yan, Lei Wang, Haigang Li, Wei Zhang, Tao Tang, Yang Wang
Brains were fixed in 4% paraformaldehyde for 24 h and cut into 3.0 μm coronal paraffin sections. For hematoxylin and eosin (H&E) staining, the rehydrated sections were immersed in hematoxylin (G1004, Servicebio, Wuhan, China) and eosin Y (G1001, Servicebio) for 5 min and 20 s, respectively. For Nissl’s staining, the slices were incubated in the toluidine blue solution (G1036, Servicebio) for 8 min. For immunofluorescent, 3% bovine serum albumin in 0.02% Triton-X100 was used for blocking. Then, primary antibodies were incubated at 4 °C for 14 h, including rabbit anti-BDNF (ab108319, 1:1000, Abcam, Cambridge, MA), rabbit anti-Tau1 (MAB3420, 1:400, Millipore, Billerica, MA), rabbit anti-GAP43 (8945S, 1:800, Cell Signaling Technology, Danvers, MA), rabbit anti-doublecortin (DCX, 4604S, 1:800, Cell Signaling Technology), mouse anti-proliferating cell nuclear antigen (PCNA, cell proliferation marker, SC-56, 1:1000, Santa Cruz Biotechnology, Dallas, TX). Cy3 or Alex Fluor 488-conjugated secondary antibodies were utilized for visualization. Negative controls were performed by omission of primary antibodies. For each measurement, three slices were stained at 0.5 mm intervals in each animal. The first slice was cut at about −3.0 mm level (relative to bregma), where the structure of the hippocampus and the edge of the wound were typical according to the standard rat stereotaxic atlas (Paxinos and Watson 2007). The experimental groups were blinded to statistic researchers.
Crab Shell Extract Improves Sperm Parameters and Antioxidant Status in Testes of Diabetic Rats
Published in Journal of Dietary Supplements, 2019
Elham Ghanbari, Mohammad Rasool Khazaei, Parinaz Ahangar, Mozafar Khazaei
The caudal part of the left epididymis from each rat was excised and placed in a petri dish containing 5 mL of Ham's F-10 solution and incubated at 37°C for 30 minutes to provide the sperm migration from the epididymis. Then the sperm suspension was transferred into a Neubauer hemocytometer chamber for the assessment of total number of sperm with light microscope (× 100) and data were presented in millions/ml of suspensions. Sperm motility was estimated through counting the number of fast progressive motile and immotile sperm and the percentages of motile and immotile sperm were obtained. Briefly, the percentage of motile sperm was recorded by placing 20 µl sperm suspension on a glass slide covered with a cover slip, and was assessed by light microscopy (magnification × 100) in at least 10 microscopic fields (Khazaei et al., 2012). The sperm suspension was used to calculate sperm viability as previously described. Briefly, 20 µl of eosin Y was added to 20 µl of sperm suspension. The percentage of sperm viability was evaluated as the number of live sperm (sperm that did not stain) over the total number of sperm.
Microwave assisted green synthesis of silver nanoparticles using leaf extract of elephantopus scaber and its environmental and biological applications
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2018
Sijo Francis, Siby Joseph, Ebey P. Koshy, Beena Mathew
The microwave assisted synthetic strategies of nanoparticles preparation were exploited in the case of AgNP-E. scaber. The XRD studies showed that the synthesized AgNP-E. scaber has a face centered cubic lattice. The spherical shape and the nano-regime size of the silver nanoparticles were well proved by TEM images. The adverse environmental impacts of organic nitrocompounds can be easily rectified by applying AgNP-E. scaber nanocatalyst. The dye eosin Y can be rapidly and successfully degraded using AgNP-E. scaber catalyst. The synthesized silver nanoparticles are beneficial to mankind by their appreciable antimicrobial and antioxidant potential. Further, we have succeeded in establishing their ability to manage skin cancer cell lines A375, in-vitro manner. The green synthetic pathway makes the AgNP-E. scaber more adequate for many therapeutic and biological applications. In-vivo studies and mores investigations are needed for the clinical adaptation of the results.