After a 1-h incubation at 65°C, the slides were stained with hematoxylin-eosin, and serial brain sections 208 μm apart between –1.60 mm and –2.10 mm relative to the bregma (3 sections/mouse) were photographed with a wide-field microscope (Nikon Eclipse 80i) equipped with a color camera (Axiocam 506c, Carl Zeiss Jena). ImageJ 1.46r software was used to outline the ipsilesional and contralesional hippocampus and hemisphere to determine the area. Since HI did not alter the hemisphere area and the contralesional hippocampus/contralesional hemisphere area ratio did not change with age, the ratio of ipsilesional to contralesional hippocampus area was used to quantify HI-induced atrophy or lack of growth of the ipsilesional hippocampus.
Eclipse 80i
Manufactured by Zeiss
Sourced in Germany
The Eclipse 80i is a microscope system designed for high-performance imaging and analysis. It features a modular design, allowing for customization to suit a variety of applications. The Eclipse 80i provides advanced optical performance, including high-resolution image capture and precise control over illumination and magnification.
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Lab products found in correlation
Lsm 510 meta, by Zeiss (4 mentions)
Lsm 780, by Zeiss (4 mentions)
Mouse anti cardiac troponin t, by Thermo Fisher Scientific (2 mentions)
Rabbit anti cardiac troponin t, by Abcam (2 mentions)
Goat anti lmna, by Santa Cruz Biotechnology (2 mentions)
Rabbit anti lmna, by Santa Cruz Biotechnology (2 mentions)
Mouse anti sarcomeric alpha actinin, by Merck Group (2 mentions)
Zen software, by Zeiss (2 mentions)
4 laser confocal microscope, by Zeiss (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Axiocam 506c, by Zeiss (1 mentions)
Lsm 510 meta confocal microscope, by Zeiss (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Low melting agarose gel, by Bio-Rad (1 mentions)
Axiovert, by Zeiss (1 mentions)
Vibratome, by Leica camera (1 mentions)
Vectashield mounting solution, by Vector Laboratories (1 mentions)
Anti tnfr1, by Abcam (1 mentions)
Mouse anti cd68, by Abcam (1 mentions)
Mouse anti map2 antibody, by Abcam (1 mentions)
17 protocols using eclipse 80i
1
Quantifying Hippocampal Atrophy in HI Injury
2
Immunostaining of Cultured Cells
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Standard immunostaining was performed. In brief, cells were grown on glass cover slips in 12-well plates before fixed with 4% paraformaldehyde. The cover slips were incubated in blocking solution, followed by incubation with the primary antibody at a 1:100 dilution overnight at 4°C. FITC-or Alexa fluor-labeled anti-rabbit or anti-mouse antibody was added to the incubation. The nuclei were stained with DAPI (Sigma). The samples were observed under a fluorescence microscope (Nikon, ECLIPSE 80i), or a ZEISS LSM 510 META confocal microscope (Carl Zeiss, Jena, Germany). The images captured by confocal microscope were using a 40× objective (Plan-Apochromat 40×/1.40 DIC M27) and processed using LSM Image browser software.
3
Immunofluorescence Analysis of Cardiac Cells
4
Fluorescence Imaging of Zebrafish Embryos
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Embryos were anesthetized in 0.168 mg/ml Tricaine methanesulfonate (Sigma, A-5040) and mounted in 0.8–1% low-melting agarose gel (Bio-Rad, 161-3111) in egg water before observation. GFP and mCherry reporter expression was observed and the various patterns were recorded under either a Nikon Eclipse 80i upright microscope or a Zeiss Axiovert inverted fluorescence imaging system, equipped with a Coolsnap monochrome camera (Photometrics, EZ). Higher magnification images were documented using a Leica SP5 TCF inverted confocal microscope.
5
Immunofluorescence Staining Protocol
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Immunofluorescence staining was performed on 5-μm frozen sections. All antibodies were diluted 1:200. No primary was used as control for all studies. Only previously validated antibodies were used. Fluorescence was measured using Nikon Eclipse80i or Zeiss 4 laser Confocal microscope using same laser output, gain, and offset for each set of antibodies tested. Images were quantified with Image J and adjusted for the area.
6
Immunohistochemistry and Optogenetic Brain Sectioning
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Mice were transcardially perfused with 4% paraformaldehyde (PFA) dissolved in PBS. Dissected brains were incubated in 4% PFA/PBS for an additional 8 h after perfusion. For immunohistochemistry, brains were dehydrated in 30% sucrose/PBS for 48 h and sectioned coronally (40 µm thickness) on a −20 °C cryostat. Brains used for optogenetic experiments were sectioned using a Vibratome (Leica) without the dehydration step. Prepared brain sections were stored in 0.02% sodium azide dissolved in PBS. For histological analysis, sections were cover-slipped with Vectashield mounting solution (Vector Laboratories) and observed under a Nikon Eclipse 80i fluorescence microscope or Carl Zeiss LSM780 confocal microscope. Through post hoc analysis of the section histology, two mice from Fig. 6d and one mouse from Fig. 6g were excluded from the behavior data due to AAV expression in the rostral ACC and PL area.
7
Immunofluorescence Staining of Neural Cells
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The immunofluorescence was performed as described previously [26 (link)] , with some modifications. Cells were fixed in cold 4 % formaldehyde for 15 min before incubation in blocking solution (5 % normal donkey serum/PBS 0.01 % Triton X-100). Cells were incubated with rabbit anti-GFAP antibody (1:500; Millipore), mouse anti-MAP2 antibody (1:500; Abcam), mouse anti-CD68 (1:200; Abcam) or anti-TNFR1 (1:300; Abcam) overnight. After washing in PBS, cells were incubated with (1:1000) anti-rabbit Alexa Fluor 488 and anti-mouse Alexa Fluor 594. DAPI was used to stain nuclei. Control cells were incubated only with secondary antibodies. Images were analysed under a fluorescence microscope (Nikon Eclipse 80i) or confocal microscope (Zeiss LSM 780).
8
Quantifying Pollen Lipid Droplets with BODIPY 505/515
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BODIPY 505/515 (4, 4-difluro-1, 3, 5, 7-tetramethyl-4-bora-3a, 4-adiaza-s-indacene; Invitrogen Molecular Probes, USA) was dissolved in anhydrous dimethyl sulfoxide (DMSO) as a stock solution at a concentration of 1.0 mg/mL. For Arabidopsis pollen staining, a final concentration of 1.0 μg/mL was used. Lipid droplets in stained pollen were observed using a Nikon Eclipse 80i microscope or a confocal microscope system (Carl Zeiss upright LSM 710 NLO). To quantify the fluorescence intensities of BODIPY 505/515 stained pollen, we first converted the images to grey scale images. The intensity of each pollen grain was then quantified using ImageJ.
9
Fungal Isolate Characterization Protocols
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All isolates were characterised following the protocols described by Leslie & Summerell (2006) and Lombard et al. (2019) using PDA, oatmeal agar (OA, recipe in Crous et al. 2019 ), synthetic nutrient-poor agar (SNA; Nirenberg 1976 ) and carnation leaf agar (CLA; Fisher et al. 1982 ). Colony morphology, pigmentation, odour and growth rates were evaluated on PDA after 7 d at 24 °C in the dark. Colour notations was done using the colour charts of Rayner (1970) . Micromorphological characters were examined using water as mounting medium on a Nikon Eclipse 80i and/or Zeiss Axioskop 2 plus with Differential Interference Contrast (DIC) optics and a Nikon AZ100 stereomicroscope, all fitted with Nikon DS-Ri2 high definition colour digital cameras to photo-document fungal structures. Measurements were taken using the Nikon software NIS-elements D v. 4.50 of at least 30 fungal structures and the 95 % confidence levels were determined for the conidial measurements with extremes given in parentheses. For all other fungal structures examined, only the extremes are presented. To facilitate the comparison of relevant micro- and macroconidial features, composite photo plates were assembled from separate photographs using PhotoShop CSS.
10
Scaffold Immunofluorescence Imaging
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Immunofluorescent stained scaffolds were examined with a Nikon Eclipse 80i epifluorescent microscope and a Zeiss confocal microscope.
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