Sperm viability was also determined by using eosin staining (Sigma Aldrich, St. Louis, MO, USA) as described previously [24 ]. Eosin penetrates non-viable, dead spermatozoa with disrupted membranes, which stain red using this technique. The percentage of normal morphology per 100 spermatozoa for each rat was assessed under light microscopy (×400; Zeiss, Munich, Germany) as described previously [22 (link),24 ]. An experienced technician blinded to the study performed all analyses.
Eosin staining
Manufactured by Merck Group
Sourced in United States, Italy
Eosin staining is a commonly used histological staining technique. It is a synthetic dye that binds to basic structures in cells, providing a pink or red coloration. Eosin staining is often used in combination with other stains, such as hematoxylin, to create a contrasting effect in microscopic samples.
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Lab products found in correlation
Haematoxylin, by Merck Group (2 mentions)
Entellan, by Merck Group (1 mentions)
Dpx mounting medium, by Merck Group (1 mentions)
Histoclear, by Merck Group (1 mentions)
Harris hematoxylin, by Bio-Optica (1 mentions)
Histoclear, by National Diagnostics (1 mentions)
Ham s f 10 solution, by Merck Group (1 mentions)
6 protocols using eosin staining
1
Sperm Viability and Morphology Assessment
2
Paraffin Tissue Staining for Histology
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Paraffin sections of 4 µm were cut, hydrated and stained with Sirius-red and Hematoxylin-Eosin (H&E) solutions. For Sirius-red staining, sections were incubated for 90 min with 1 g/L Sirius-red F3B in picric acid (both Klinipath) and subsequently incubated for 10 min with 0.01 M HCl. H&E staining was performed by 5 min incubation with Hematoxylin solution (Mayer, Merck) followed by a 10 min wash with tap water and a 30 seconds Eosin staining (Sigma). After the staining slides were dehydrated and mounted with Entellan (Merck KGaA).
3
Cell Morphology Assessment via Staining
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Cells were seeded into 6-well culture plates at 1 × 105 cells/ml ratio in extraction medium containing each sample. Cells were maintained at +37°C ± 1 in a humidified 5% CO2 atmosphere for 72 h in order to perform haematoxylin/eosin staining. Briefly, media were removed from each well and cells were washed with PBS and fixed in methanol; 1% haematoxylin (Sigma-Aldrich) solution was added, followed by PBS washings and 1% eosin staining (Sigma-Aldrich). Cell morphology was evaluated by using an inverted microscope supplied with a camera (Leica).
4
Histological Analysis of Adipose Tissues
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Five μm-thick paraffin-embedded sections of formalin-fixed liver, ingWAT and BAT samples were deparaffinised for 20 min using Histo-Clear (HS-200, National Diagnostics) and rehydrated through graded ethanol solutions (100-70%) to distilled water. Sections were incubated with Harris Hematoxylin (Bio-Optica, Milano, Italy) for 15 min, followed by eosin staining (Sigma Aldrich) during 15 min. Next, sections were rinsed in running tap water and differentiated with 0.5% (v/v) HCl. Finally, samples were dehydrated through graded ethanol solutions (70-100%), cleared with Histo-Clear and mounted with DPX mounting medium (06522, Sigma-Aldrich). Five to ten random images per sample were blindly taken using a Leica DM750 microscope. Number of adipocytes per field were counted using ImageJ software https://imagej.nih.gov/ij .
5
Epididymal Sperm Extraction and Evaluation
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Epididymal spermatozoa were collected by dissecting the caudal part of the left epididymis. The spermatozoa were separated from the epididymal tubules by chopping the caudal part of the epididymis in 5 mL of Ham's F-10 solution (Sigma-Aldrich). The solution was incubated for 10 minutes at 37℃ to release the spermatozoa into the medium. After pipetting, 100 µL of the sperm suspension was diluted with 900 µL of saline. The diluted sperm suspension was transferred into a Neubauer hemocytometer chamber and the sperm heads were counted [29 30 (link)]; data were expressed as the total number of sperm per milliliter.
In this study, the percentage of motility was evaluated for all animals. Briefly, the spermatozoa were classified as motile or immotile. Sperm viability was also determined by using Eosin staining (Sigma-Aldrich) as described previously [29 ]. Eosin penetrated non-viable, dead spermatozoa with disrupted membranes, which stained red. The percentage of normal morphology of 100 spermatozoa per rat was assessed by light microscopy (400×, Zeiss, Munich, Germany) as described previously [29 31 (link)]. One experienced technician blinded to the study performed all analyses.
In this study, the percentage of motility was evaluated for all animals. Briefly, the spermatozoa were classified as motile or immotile. Sperm viability was also determined by using Eosin staining (Sigma-Aldrich) as described previously [29 ]. Eosin penetrated non-viable, dead spermatozoa with disrupted membranes, which stained red. The percentage of normal morphology of 100 spermatozoa per rat was assessed by light microscopy (400×, Zeiss, Munich, Germany) as described previously [29 31 (link)]. One experienced technician blinded to the study performed all analyses.
6
Cell Viability in Herbal Extract Media
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Cells were seeded into 6-well culture plates at 1 × 105 cells/ml ratio in seven different media: negative control (I), pin extract (II), ZDBC extract (III), ZDEC extract (IV), HDP extract (V), 5% phenol solution (VI), and reagent control (VII).
Cells were maintained at +37°C ± 1 in a humidified 5% CO2 atmosphere and monitored daily by using an inverted microscope for 72 h in order to evaluate cell morphology and monolayer integrity. After 72 h incubation, haematoxylin/eosin staining was performed. Briefly, media was removed from each well, and cells were washed with PBS and fixed in methanol; 1% haematoxylin (Sigma-Aldrich) solution was added, followed by PBS washing, and 1% eosin staining (Sigma-Aldrich). Cell morphology was evaluated by using an inverted microscope supplied with camera (Leica).
Cells were maintained at +37°C ± 1 in a humidified 5% CO2 atmosphere and monitored daily by using an inverted microscope for 72 h in order to evaluate cell morphology and monolayer integrity. After 72 h incubation, haematoxylin/eosin staining was performed. Briefly, media was removed from each well, and cells were washed with PBS and fixed in methanol; 1% haematoxylin (Sigma-Aldrich) solution was added, followed by PBS washing, and 1% eosin staining (Sigma-Aldrich). Cell morphology was evaluated by using an inverted microscope supplied with camera (Leica).
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