Sections were stained with H&E, and their images were taken under a light microscope. The whole area of each hemi-calvarium was imaged using Olympus DSU microscope within 4–5 image fields per section and these images were tiled. The margin of eosinophilic bone structures including frontal and parietal bones was then outlined using the free hand selection in the Image-J program, and the area was calculated and determined as the formed bone area. A total of 5 hemi-calvaria samples were used per treatment group, and 5 sections per sample were used for quantification. The values are shown as the mean (mm2)±S.D. (n=5) with statistical analysis.
Dsu microscope
Manufactured by Olympus
Sourced in Japan
The DSU microscope is a high-performance digital scanning unit designed for precise and efficient imaging. It is a core component of Olympus' advanced microscopy systems, providing reliable and consistent results for a wide range of applications.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Ghost fixable viability dye, by Cytek Biosciences (1 mentions)
Laminin, by Merck Group (1 mentions)
Ab14613, by Abcam (1 mentions)
Hoechst, by Merck Group (1 mentions)
Goat anti rabbitalexa647, by Thermo Fisher Scientific (1 mentions)
Clone m2, by Merck Group (1 mentions)
Poly l ornithine, by Merck Group (1 mentions)
Low melting point agarose, by Thermo Fisher Scientific (1 mentions)
Fluoromount, by Merck Group (1 mentions)
10 protocols using dsu microscope
1
Quantifying Bone Formation in Hemi-Calvaria
2
Maintaining Transgenic Hydra Cultures
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Transgenic H. vulgaris were maintained according to standard protocols provided by the Steele lab at UC Irvine43 . Hydra were raised in Hydra medium43 at 18°C in the dark, fed 1×/week with artemia, and cleaned 3×/week. For imaging on the PIC, a specimen that was starved for at least 3 days was mounted on the chip following the same procedure as for planarians. Specimens were imaged on the Olympus DSU microscope.
3
GUS Activity Histochemical Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Histochemical analysis of the GUS activity was conducted as described (Zhou et al., 2015 (link)). Briefly, the germinating seeds, seedlings or other plant tissues were placed in a solution containing 0.2M Sodium Phosphate pH7.0, 2 mM K3 [Fe(CN) 6], 2 mM K4[Fe(CN) 6], 2 mM 5-bromo-4-chloro-3-indolyl-D-glucuronide (X-Gluc), and 10% methanol) and vacuum infiltrated for about 30 min before incubation at 37°C for one or two days. The stained materials were rinsed with a series of ethanol dilutions and subsequently incubated in 70% ethanol solution overnight to remove chlorophylls. The plants were then mounted on microscope slides and examined using an Olympus DSU microscope.
4
Histomorphometric Analysis of Calvaria Bone
5
Eosinophil Differentiation from Murine Bone Marrow
6
Immunocytochemical Analysis of PDE4D Localization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To investigate PDE4D protein localization, HT22 cells were seeded on 12 mm glass coverslips (VWR, 631–1577) coated with 100 μg/mL Poly-l -Ornithine (Sigma, P4957) and 1 μg/mL laminin (Sigma, L2020) and grown for 24 h. After fixation in 4% paraformaldehyde, cells were permeabilized using 0.1% Triton X-100. After blocking with 10% BSA for 1 h, cells were incubated with rabbit anti-PDE4D (1:250; ab14613, Abcam) overnight at 4 °C. Then, cells were incubated with goat anti-rabbit Alexa647 (1:250; Invitrogen) for 1 h at room temperature. Finally, nuclei were counterstained with Hoechst (1:500; Sigma).
For immunocytochemistry following transfection experiments in HT22, the same protocol was used except for the antibodies used. Mouse anti-FLAG primary antibodies (1:1000; M2 clone, Sigma-Aldrich) and donkey anti-mouse Alexa488-conjugated secondary antibody (1:250; Invitrogen) were used to determine which cells were successfully transfected and expressed the FLAG-encoding PX458 plasmid. PDE4D localization was imaged after mounting the coverslips on microscope glasses using a disk spinning unit (DSU) microscope (Olympus). Morphology assessment of transfected, FLAG-positive HT22 cells was performed as described above.
For immunocytochemistry following transfection experiments in HT22, the same protocol was used except for the antibodies used. Mouse anti-FLAG primary antibodies (1:1000; M2 clone, Sigma-Aldrich) and donkey anti-mouse Alexa488-conjugated secondary antibody (1:250; Invitrogen) were used to determine which cells were successfully transfected and expressed the FLAG-encoding PX458 plasmid. PDE4D localization was imaged after mounting the coverslips on microscope glasses using a disk spinning unit (DSU) microscope (Olympus). Morphology assessment of transfected, FLAG-positive HT22 cells was performed as described above.
7
Heat-Shock Induced Testis Cytoskeleton Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Crosses were started to generate males of the appropriate genotypes, and their progeny were heat-shocked using the 24 hour collection protocol. Testes were dissected from adult males within 24 hours of eclosion, fixed in 1× PBS + 4% paraformaldehyde and then stained with DAPI and phalloidin coupled to FITC or rhodamine. Testes were mounted in 50% glycerol + antifade and examined with an Olympus DSU microscope using Slidebook 5.0 software. When examining sperm head location, we counted a sperm head as displaced caudally from the bouquet if it was separated by the length of at least one sperm head. (Most displaced sperm heads showed much greater separation than this.) To facilitate scoring sperm heads in individual cysts the testis sheath was torn with forceps and, after placing a coverslip on the sample, it was tapped gently to release and spread the contents. Meiotic figures were scored in intact testes. A 2×2 contingency test was used to compare the number of testes found in wildtype vs. lok males. The Mann-Whitney test was used to compare the number of MII bridges found in wildtype vs. lok testes.
8
Quantitative Analysis of Calvarium Bone Formation
9
Planarian Anesthesia and Immobilization Protocols
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Planarians were anesthetized with 0.2% chloretone (5 minutes exposure), 0.08% chloretone (2 hours exposure), or 3% ethanol (1 hour exposure). Worms were removed from 0.2% chloretone immediately prior to imaging but kept in 0.08% chloretone and 3% ethanol during imaging. For the escape time experiments (Fig. 1 (C) ), anesthetized planarians were observed using the Olympus DSU microscope at 4×, and the time for worms to move completely out of view was measured using a stopwatch. For physical immobilization, planarians were placed in small glass-bottom dishes (MatTek Corporation, Ashland, MA) in 2% low melting point agarose (Life Technologies) that was still liquid but cool enough not to injure the worms. The dishes were cooled on custom Peltier plates while the agarose solidified to minimize specimen movement. Planarians were imaged after the agarose had solidified completely, which typically was 30 minutes or longer after embedding.
10
Raichu-Rac1 Biosensor FRET Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Raichu-Rac1 biosensor was used for FRET experiments (Itoh et al., 2002) . The Rac1 activation induces a conformational change of the probe resulting in energy transfer from CFP emission energy to YFP excitation energy, which is measured as FRET efficiency (Itoh et al., 2002) . Cells (2 × 10 6 ) were transfected with the biosensor plasmid (4 μg) by electroporation (Amaxa) and plated on laminin coverslips. After 24 h of expression, recombinant Reelin (1 μg/ml) o vehicle was added for 10 and 20 min. The reaction was stopped washing the cells with chilled PBS and fixed with 4% paraformaldehyde/Sucrose for 10 min. After washing coverslips were mounted on glass slides with Fluoromount™.
(Sigma-Aldrich). FRET images were acquired in a DSU microscope (Olympus). For emission ratio imaging, the following filter set were used: CFP: 490/500 HQ, DM505, 515/560HQ; FRET: 490/500 HQ, DM505, 527/565HQ. FRET map images were calculated by Ratio imaging using ImageJ software using the following formula: (CFP emission/YFP emission, both exited at the same excitation wavelength (CFP). Before FRET map calculation, images were background subtracted and corrected for alignment channel. An average of 30 cells per condition was considered for FRET analyses.
(Sigma-Aldrich). FRET images were acquired in a DSU microscope (Olympus). For emission ratio imaging, the following filter set were used: CFP: 490/500 HQ, DM505, 515/560HQ; FRET: 490/500 HQ, DM505, 527/565HQ. FRET map images were calculated by Ratio imaging using ImageJ software using the following formula: (CFP emission/YFP emission, both exited at the same excitation wavelength (CFP). Before FRET map calculation, images were background subtracted and corrected for alignment channel. An average of 30 cells per condition was considered for FRET analyses.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!