To obtain the histological sections, the contaminated and uncontaminated seminal vesicles were dissected and fixed for 2 h in 2.5% glutaraldehyde solution in 0.1 M phosphate buffer, pH 7.2 at 4 °C. The material was washed for 2 h in the same buffer, post fixed in 1% osmium tetroxide for 2 h and dehydrated in alcohol solutions of increasing concentrations: 30, 50, 70, 90 and 100%. The material was immersed in two 4-h baths each at room temperature, the first with a mixture of Historesine (Leica Historesin, Heidelberg, Germany) and alcohol (1:1), and the second with pure Historesin. For inclusion, the seminal vesicles were immersed in Historesin with a catalyst in silicone moulds, which were placed in Petri dishes and transferred to an oven at 58 °C for 24 h. Semithin sections (2 µm) were obtained with a microtome Leica RM 2155 (Leica Corporation, Wetzlar, Germany) with glass knives. These were transferred to histological slides stained with Harris haematoxylin for 15 min, washed in running water for 10 min, stained with eosin for 1 min and rapidly rinsed in tap water. For the detection of neutral polysaccharides some slides were submitted to the periodic acid-Schiff (PAS) reaction. All observations and photographs were made using an Olympus BX-60 microscope.
Historesin
Manufactured by Leica camera
Sourced in Germany, France
Historesin is a water-miscible, glycol-based resin used for embedding biological samples for light microscopy. It provides good preservation of cellular and tissue morphology, allowing for thin sectioning and subsequent staining and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Microtome, by Leica camera (4 mentions)
Rm2245, by Leica camera (3 mentions)
Bx60 microscope, by Olympus (3 mentions)
Entellan, by Merck Group (3 mentions)
Leica microtome, by Leica camera (3 mentions)
Jung rm2055 microtome, by Leica camera (3 mentions)
Rm 2155, by Leica camera (3 mentions)
Axioplan 2 microscope, by Nikon (2 mentions)
Dxm1200f, by Nikon (2 mentions)
Bx51 microscope, by Olympus (2 mentions)
Histosec, by Merck Group (2 mentions)
Em scd 500, by Leica camera (2 mentions)
Jsm 6390lv, by JEOL (2 mentions)
Axioskop 2 plus, by Zeiss (2 mentions)
Image plus 2, by Motic (2 mentions)
Rm2135, by Leica camera (1 mentions)
Bx51 photomicroscope, by Olympus (1 mentions)
Leica historesin, by Leica camera (1 mentions)
Axiocam 105 color, by Zeiss (1 mentions)
Digital camera, by Olympus (1 mentions)
79 protocols using historesin
1
Histological Analysis of Seminal Vesicles
2
Ovary Dissection and Histological Analysis
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Insects were cryoanesthetized at −4 °C for 90 s, and their ovaries were dissected in 125 mM NaCl. Five pairs of ovaries were transferred to 4% paraformaldehyde in 0.1 M phosphate buffered saline (PBS), pH 7.2. Then, samples were washed in the same buffer, dehydrated in a graded ethanol series (70, 80, 90, and 95%), and embedded in historesin (Leica). Sections of 2 μm, obtained using a rotary microtome with a glass knife, were stained with hematoxylin and eosin and analyzed under a light microscope.
3
Retinal Histology and Thickness Analysis
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Fixed eyes were embedded in historesin (Leica, Nanterre, France) to facilitate the cutting of 5-μm-thick sections through the detached area. Sections were stained with 1% toluidine blue solution. Sections were observed on a Leitz microscope. Sections with the most detached retinal area were photographed (20×) with a Leica camera for measurements (n = 5 WT mice, n = 5 TG mice, n = 5 BSS-treated rats, and n = 5 TF-treated rats). Retinal thicknesses [from the OSs to the inner part of the ganglion cell layer (GCL) nuclei], the ONL, and the outer and inner segments were measured every 100 μm (mice) and 300 μm (rats) using ImageJ software by two different blind experimenters. Retinas were considered detached when an empty space was observed between the RPE and the OSs and attached when any signs of histological disturbance were observed. The retinal thickness ratio was calculated as the thickness in the detached retina/the thickness in the attached retina. For the ONL and segment thickness ratio, layer measurements were reported as the total retinal thickness of the corresponding retinal area using the formula (ONL detached/total retina detached)/(ONL attached/total retina attached).
4
Fixation and Histological Analysis of Testes
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Six animals of different exposed groups (24) were perfused with glutaraldehyde 4% and paraformaldehyde 4% in sodium cacodylate buffer 0.1 M (pH 7.2) for 25–30 min. Testis was removed, post-fixed in 1% osmium in the same buffer solution, overnight, and then weighed. Historesin (Leica) embedded testes fragments were sectioned at 3 µm thickness and stained with toluidine blue in 1% sodium borate (TB) for structural and morphometrical evaluations.
5
Histological analysis of fish tissues
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Kidney and gill samples fixed for 72 h in 10% chilled formalin were removed from the formalin and dehydrated in increasing gradients of ethanol (50%, 60%, 70%, 85%, 95%, and 100%). The dehydrated tissue was embedded in hydroxyethyl methacrylate (Historesin, Leica, Germany) and sectioned into a series of ≈8–10 sections per sample (section thickness: ≈5 µm), mounted on slides and colored using Hematoxylin-eosin (H/E). The gill sections were stained with Periodic Acid Schiff (PAS) [48 ], for the identification of mucus cells. The Von Kossa staining method [49 (link)] was used for the identification of chloride cells with calcium in the gill sections. Sections were stained with 1% silver nitrate for 20 min under the UV light, washed in water, immersed in 5% thiosulphate for 5 min to remove excess silver nitrate on the slide, washed in water, and stained with Hematoxylin for 5 min. Microscopic analyses were done using a Zeiss Axioplan 2 microscope mounted with a Nikon digital camera DXM 1200F (Oberkochen, Germany). In each case, nine representative fish per treatment were examined.
6
Neurodegenerative Index Quantification
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Ten flies of each treatment group, at the best performed concentration in previous experiments were used. All flies were analyzed at 10 d.p.e. and elav-Gal4 was used as a control. The flies had its head transferred to 4% formaldehyde in sodium phosphate buffer 0.1 M pH 7.2 for 16 h at 4 °C. The samples were then dehydrated in a graded ethanol series (70, 80, 90, and 95%) and transferred to methanol for 16 h at 4 °C. After, they were embedded in HistoResin (Leica) and 2 µm thick slices were stained with hematoxylin and eosin, analyzed and photographed with a light photomicroscope. Neuropile images were used to calculate the neurodegenerative index—as normal to low, moderate, or severe—according to vacuolar lesions90 (link).
7
Histological Analysis of Liver Tissue
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The liver tissues were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer for 24 h. Blocks of liver tissue were taken from the median lobe and prepared for embedding in glycol methacrylate (Historesin, Leica) using standard techniques. Subsequently, sections of 5 μm thicknesses were obtained and stained with Eosin and Hematoxylin dyes for microscopic assessment. The histological sections were examined under a light microscope using a 121 intersection grid placed at the ocular lens, coupled to a 100× objective. The hepatic parenchyma was classified as one of the following: hepatocyte cytoplasm or degenerative hepatocytes, central vein, portal space, and inflammatory infiltrate.
8
Multi-staining Analysis of Biological Samples
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The samples were fixed in 2.5% paraformaldehyde in 0.2 M (pH 7.2) phosphate buffer overnight. Subsequently, the samples were dehydrated in increasing series of ethanol aqueous solutions [17 (link)]. After dehydration, the samples were infiltrated with Historesin (Leica Historesin). Sections (5 μm) were obtained using a manual rotation microtome (Leica model RM 2135) with tungsten blades. The sections were stained with Toluidine Blue O (TB-O) 0.5%, pH 3.0, to identify acid polysaccharides [18 ]; Periodic Acid-Schiff (PAS) to identify neutral polysaccharides [19 ]; and Coomassie Brilliant Blue (CBB) 0.4% in Clarke's solution to identify proteins [19 ]. Some sections were double-stained with PAS + CBB [20 ]. Sections were analyzed with a camera (Olympus® DP 71) attached to a microscope (Olympus® BX-40).
9
Histological Analysis of Fish Gonads
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Larvae and juvenile gonads from fish at different phases of development were dissected and fixed in Bouin’s solution for 24 h at room temperature, subsequently dehydrated, embedded in paraffin or historesin (Leica), and then serially sectioned at 3 to 5 μm thickness. The sections were counterstained with hematoxylin & eosin.
10
Morphoanatomical Analysis of P. pubescens Seeds
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For the morphoanatomical analyses, 2 cm2 samples were obtained from the endosperm region of P. pubescens seeds from each color class subsequently fixed in Karnovsky’s solution [43 ] for 24 h. The samples were then pre-washed in a phosphate buffer (0.1 M, pH 7.2) and dehydrated in an increasing ethanol series (30% to 100%) and, finally, pre-infiltrated and infiltrated in Historesin (Leica, Wetzlar, Germany), as per the manufacturer’s recommendations. The samples were then sectioned transversely using a rotating microtome (Model 1508R, Logen Scientific, Shanghai, China), obtaining 5 μm thick sections and stained with toluidine blue-polychromatic staining (0.05% 0.1 M phosphate buffer, pH 6, 8) [44 (link)]. Starch was detected by histochemical staining with a 10 g L−1 lugol solution [45 ]. Total protein detection was performed through Xylidine ponceau (XP) staining [46 ]. Images were obtained using an Olympus microscope (BX61, Tokyo, Japan), coupled to a DP-72 camera, using the brightfield option.
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