Embryos were fixed in 4% (w/v) paraformaldehyde (in phosphate-buffered saline) and either dissected or processed for cryostat sectioning. Tissue was stained by in situ hybridisation (Myat et al., 1996 (link)) with digoxygenin- or fluorescein-labelled (Roche) riboprobes for: Atoh1 (Wilson and Wingate, 2006 (link)), Otx2 (Millet et al., 1996 (link)), Ptf1a (ChEST1028o4), Lhx9 (Alessio Delogu, King’s College London, UK), Gdf7 (Broom et al., 2012 (link)), Gbx2 (Kiecker and Lumsden, 2004 (link)), Fgf8 and Sprouty2 (Chambers et al., 2000 (link)); and mouse probes for: Atoh1 (Albert Basson, King’s College London, UK), Lhx9 and Tbr1 (Alessio Delogu, King’s College London, UK). Following in situ hybridisation, GFP signal was amplified immunohistochemically with an anti-GFP antibody (IgG 1:100, Invitrogen) and mitotic cells were detected with an anti-phospho-histone H3 antibody (1:100, NEB). Some whole-mounts were further processed for vibratome sectioning. Digital brightfield and fluorescence images were acquired on either stereo (Leica MZFLIII) or compound (Nikon Elipse80i) microscopes equipped with epifluorescence or by laser scanning confocal microscopy (Olympus AX70).
Elipse 80i
Manufactured by Nikon
Sourced in Japan
The ELIPSE 80i is a high-performance laboratory microscope designed for a variety of applications. It features a sturdy, ergonomic design and offers a range of optical configurations to meet the needs of various research and analysis tasks.
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Lab products found in correlation
8 protocols using elipse 80i
1
In situ hybridization analysis of embryonic gene expression
2
Quantitative Analysis of Gonadal Receptors
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The FSHR, LHR, GnRH and GnRHR labeling was examined by a microscope (Nikon ELIPSE 80i, Tokyo, Japan). Three stained sections of each sample were randomly selected, and three visual fields of each stained section were randomly selected for observation and photography. To estimate the amount of cell staining, the pictures were analyzed using an image analysis software (Image Pro-Plus 6.0, Media Cybernetics, Silver Spring, MD, USA) [58 (link)]. Total cross-sectional IOD was acquired, which was used to compare the FSHR, LHR, GnRHR and GnRH staining intensity between control and ZEA treatments. The IOD of each sample is the average of three stained sections.
3
Histomorphometric Analysis of Duodenal Tissue
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The fixed duodenum was embedded in a fully automatic rotary paraffin slicer (HM355S, Burton International Trading Co., Ltd, China), sliced to a 5 μm thickness using a microtome device. After that, the samples were dyed by an automatic dyeing machine (A81500101, Thermo Scientific, UK) and sealed with neutral resin. The morphology of the duodenum was observed and photographed by microscope (Nikon elipse 80i, Japan). For each sample, 10 well-oriented villi and corresponding crypts were measured by an image analyzer (Lucia software, zadrahau), and the ratio of villi to crypts was calculated.
4
Quantifying GHR Expression in Uterine Tissue
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Histological sections of the uteri were observed using a microscope (Nikon ELIPSE 80i, Tokyo, Japan) at magnifications of ×40, ×100, and ×200. To evaluate the amount of cell staining and quantity of the target antigen of GHR, the images were analyzed using image analysis software (Image Pro-Plus 6.0, Media Cybernetics, Sliver Spring, MD, USA). This yielded values of the total cross-sectional integrated optical density (IOD) [27 (link)], which were used to compare the amount of GHR staining in different parts of the uteri of GHR in the different treatments. We examined at least five stained sections, which were randomly selected from the 10 piglets in each group.
5
Microscopic Evaluation of Uterine Antigens
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Histological sections of the uteri were observed using a microscope (Nikon ELIPSE 80i, Tokyo, Japan) at magnifications of ×200. To evaluate the amount of cell staining and quantity of the target antigen of PCNA, BAX, and BCL-2, the images were analyzed using image analysis software (Image Pro-Plus 6.0, Media Cybernetics, Sliver Spring, MD, USA). This yielded values of the total cross-sectional integrated optical density (IOD) [51 (link)], which were used to compare the staining amount in the different treatments. We examined at least five stained sections, which were randomly selected from the 10 piglets in each group.
6
Visualizing Axon Development in Chick and Mouse Embryos
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Electroporated chick embryos and control chick and CD1 mouse embryos were fixed in 4% (w/v) paraformaldehyde (in phosphate-buffered saline) and either dissected or processed for cryostat sectioning. Tissue was stained by in situ hybridisation (Myat et al., 1996) with digoxygenin-labelled (Roche Diagnostics Limited, Burgess Hill, West Sussex, UK) riboprobes for (mouse) Lhx9, Tbr1 (Alessio Delogu, Kings College London, UK) or (chick), Lhx9 (Alessio Delogu), Tbr1[30 (link)] and Pax6[36 (link)]. Following in situ hybridisation tissue was processed for fluorescence immunohistochemistry using an anti-GFP antibody (IgG 1:100, Invitrogen). Digital images were acquired on either stereo (Leica MZFLIII) or compound (Nikon Elipse80i) microscopes equipped with epifluorescence or by laser scanning confocal microscopy (Olympus AX70). Axon initial segment orientation was measured in ImageJ.
7
Uterine Immunohistochemistry Protocol
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The histological sections of the uterus were dewaxed and rehydrated for immunohistochemical chemical analysis according to standard immunohistochemistry protocols [55 (link),56 (link),57 (link)]. Antigen retrieval was performed in sodium citrate buffer (0.01 mol/L, pH 6.0) using a microwave unit for 20 min at full power. The sections were then washed with phosphate-buffered saline (PBS) (0.01 mol/L, pH 7.2).
Five stained sections randomly selected from ten gilts in each group were examined under a microscope (ELIPSE 80i; Nikon, Tokyo, Japan). The amount of cell staining and quantity of the target antigen of ER-α, ER-β and PR were evaluated and analyzed by Image Pro-Plus 6.0 (Media Cybernetics, Silver Spring, MD, USA). This yielded value of the total cross-sectional integrated optical density (IOD) [57 (link)] was used to compare the staining amount in the different treatments.
Five stained sections randomly selected from ten gilts in each group were examined under a microscope (ELIPSE 80i; Nikon, Tokyo, Japan). The amount of cell staining and quantity of the target antigen of ER-α, ER-β and PR were evaluated and analyzed by Image Pro-Plus 6.0 (Media Cybernetics, Silver Spring, MD, USA). This yielded value of the total cross-sectional integrated optical density (IOD) [57 (link)] was used to compare the staining amount in the different treatments.
8
Microscopic Analysis of Ovarian Histology
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The histological sections of the ovaries were observed under a microscope (ELIPSE 80i; Nikon, Tokyo, Japan). Five stained sections randomly selected from ten gilts in each group were examined. To evaluate the amount of cell staining, the images were analyzed using an image analysis software (Image Pro-Plus 6.0; Media Cybernetics, Silver Spring, MD, USA). To assess the expression intensity of positive substance, we used an image analysis software (Image Pro-Plus 6.0; Media Cybernetics) to obtain the total cross-sectional IOD.
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