Purified recombinant proteins and BPEVdsRNA were diluted in buffer C (5 mM HEPES–NaOH pH 7.5, 50 mM NaCl, 5 mM MgCl2, 150 nM MDA5, 8.4 nM dsRNA and 30 nM LGP2). Mixtures were incubated at 37°C. Then, RNA/protein samples were fixed with 0.05% glutaraldehyde (Nacalai Tesque, Japan), placed on 10 mM Spermidine treated mica for adhesion and dried with nitrogen gas before imaging.
Glutaraldehyde
Manufactured by Nacalai Tesque
Sourced in Japan
Glutaraldehyde is a chemical compound used as a fixative and cross-linking agent in various laboratory applications. It is a colorless, oily liquid with a pungent odor. Glutaraldehyde is commonly used for the preservation and stabilization of biological samples, such as tissues and cells, prior to analysis or further processing.
Automatically generated - may contain errors
Lab products found in correlation
Paraformaldehyde (pfa), by Nacalai Tesque (4 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Glycine, by Fujifilm (2 mentions)
Paraformaldehyde (pfa), by Merck Group (2 mentions)
Axiocam mrm, by Zeiss (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 goat anti rabbit antibody, by Thermo Fisher Scientific (1 mentions)
Axio imager z2, by Zeiss (1 mentions)
Axiovision software, by Zeiss (1 mentions)
Alexa fluor 488 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Potassium ferricyanide, by Fujifilm (1 mentions)
Magnesium chloride, by Fujifilm (1 mentions)
Potassium ferrocyanide, by Fujifilm (1 mentions)
Formaldehyde, by Nacalai Tesque (1 mentions)
Osmium tetroxide, by Electron Microscopy Sciences (1 mentions)
Quantax 400, by Bruker (1 mentions)
Formalin, by Fujifilm (1 mentions)
Bouin s solution, by Fujifilm (1 mentions)
Alexa fluor 488 labeled phalloidin, by Thermo Fisher Scientific (1 mentions)
Fructose, by Fujifilm (1 mentions)
30 protocols using glutaraldehyde
1
Imaging MDA5-dsRNA Complexes
2
Mitochondrial Ultrastructure of Osteoclasts
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BMMs (1*106 cells/well) were cultured in a medium containing M-CSF and RANKL and treated using OMVs for 3 days. After harvesting, cells were centrifuged at 4°C for 5 min at 2,000 rpm. About 2.5% glutaraldehyde (Nacalai Tesque, Tokyo) was added to fix the cells at RT for 1 h and then left at 4°C for 3 h. After aspirating the fixative, cells were suspended in PBS, and TEM was used to observe the ultrastructure of mitochondria.
3
Immunofluorescent Localization of Plasmodium yoelii Proteins
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Schizont-rich whole blood was obtained from P. yoelii infected mouse tail and prepared air-dried thin smears on glass slides. The smears were fixed in 4% paraformaldehyde containing 0.0075% glutaraldehyde (Nacalai Tesque) in PBS at room temperature (RT) for 15 min, rinsed with 50 mM glycine (Wako) in PBS. Samples were permeabilized with 0.1% Triton X–100 (Calbiochem) in PBS for 10 min, then blocked with 3% BSA (Sigma) in PBS at RT for 30 min. Next, samples were immunostained with primary antibodies using mouse anti–PyEBL [26 (link)] (final 1:500) and Rabbit anti–PyAMA1 [45 (link)] (a gift from Takafumi Tsuboi, final concentration 1:500) at 37°C for 1 h. This was followed by 3 washes with PBS then incubation with Alexa Fluor 488 goat anti–mouse and Alexa Fluor 594 goat anti–rabbit antibodies (Invitrogen; final 1:1000) in 3% BSA in PBS at 37°C for 30 min. Parasite nuclei were stained with 4’, 6-diamidino-2-phenylindole (DAPI; Invitrogen, final 0.2 μg/mL). Stained parasites were mounted with Prolong Gold antifade reagent (Invitrogen). Slides were visualized using a fluorescence microscope (Axio imager Z2; Carl Zeiss) with 100x oil objective lens (NA 1.4, Carl Zeiss). Images were captured using a CCD camera (AxioCam MRm; Carl Zeiss) and imaged using AxioVision software (Carl Zeiss). Mann-Whitney U tests were performed using Graphpad Prism software (v6.01).
4
High-Resolution Ultrastructural Imaging of Ciona Eggs
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Dechorionated Ciona eggs/embryos were fixed with 2.5% glutaraldehyde (Nacalai Tesque) containing Fix solution 1 for 1 h at room temperature. The fixed specimens were treated with ethanol up series (35%, 70%, and 100%) and stored at − 30 °C until further use. The specimens were post-fixed in 2.0% osmium tetroxide (Nisshin-EM, Tokyo, Japan) and 1.5% potassium ferrocyanide in artificial sea water (420 mM NaCl, 9 mM KCl, 10 mM CaCl2, 24.5 mM MgSO4, 2.15 mM NaHCO3, HEPES [pH = 8.0]) for 1 h at 4 °C. The post-fixed specimens were then treated with 1% thiocarbohydrazide (Tokyo Chemical Industry, Tokyo, Japan) for 20 min at room temperature. The specimens were post-fixed again in 2.0% osmium tetroxide for 30 min at room temperature. The specimens were stained with 3% Ti-blue (Nisshin-EM) for 16 h at 4 °C. Next, the specimens were stained with 0.03 M aspartic acid and 0.02 M lead nitrate for 30 min at 60 °C. After dehydration with EtOH up series and acetone, the specimens were embedded in epoxy resin (CLEARPOXY Resin, Sankei Co., Ltd, Tokyo, Japan). The specimens were observed using FIB-SEM (NX5000; Hitachi, Tokyo, Japan). More than 40 eggs were observed in the present study.
5
Senescence-Associated β-Galactosidase Assay
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hiHepPCs in monolayer cultures were fixed with PBS containing 2% formaldehyde (Nacalai Tesque) and 0.2% glutaraldehyde (Nacalai Tesque), and senescence-associated β-galactosidase activities in hiHepPCs were analyzed as described previously45 (link). Briefly, the fixed cells were washed in PBS and incubated for 16 h at 37 °C with a mixture of 0.5 mg mL–1 X-gal (Anatrace) and 40 mM citric acid/sodium phosphate buffer (pH 6.0) containing 5 mM potassium ferricyanide (Wako), 5 mM potassium ferrocyanide (Wako), and 2 mM magnesium chloride (Wako).
6
Lateral Wall Wholemount SEM Protocol
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Wholemount preparations of the lateral wall of the LVs were fixed with 2% PFA and 2.5% glutaraldehyde (Nacalai Tesque) in PB at RT for 1 h, washed with PB, postfixed with 1% osmium tetroxide (Electron Microscopy Sciences) in PB for 45 min at RT, rinsed with deionised water and dehydrated first in ethanol then with CO2 using the critical point drying method. The samples were coated with gold/palladium alloy by sputter coating. The surface of the lateral wall was examined under a Hitachi S-4800 scanning electron microscope using Quantax 400 software (Bruker) for image acquisition.
7
Tissue Preparation and Staining Techniques
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Whole larvae were fixed overnight in a solution of 4% paraformaldehyde (PFA) and 5%
sucrose in 0.1 M phosphate buffer at 4°C, and embedded in paraffin. Adult and juvenile
zebrafish were euthanized by immersion in ice-cold water or 1:1,000 dilution of
2-phenoxyethanol. After the euthanasia, adult and juvenile zebrafish were decapitated, and
the heads were fixed overnight in Bouin’s solution (Wako Pure Chemical Industries, Ltd.,
Osaka, Japan) at 4°C, and thereafter embedded in paraffin. The paraffin blocks were cut in
5 µm thick sections, and stained with hematoxylin and eosin
(H&E).
The eyes of rats were fixed overnight in a solution of 10% formalin (Wako Pure Chemical
Industries, Ltd.): 25% glutaraldehyde (Nacalai Tesque, Inc., Kyoto, Japan) (9:1) at 4°C,
and thereafter embedded in paraffin. H&E stained specimens were prepared under routine
method.
sucrose in 0.1 M phosphate buffer at 4°C, and embedded in paraffin. Adult and juvenile
zebrafish were euthanized by immersion in ice-cold water or 1:1,000 dilution of
2-phenoxyethanol. After the euthanasia, adult and juvenile zebrafish were decapitated, and
the heads were fixed overnight in Bouin’s solution (Wako Pure Chemical Industries, Ltd.,
Osaka, Japan) at 4°C, and thereafter embedded in paraffin. The paraffin blocks were cut in
5 µm thick sections, and stained with hematoxylin and eosin
(H&E).
The eyes of rats were fixed overnight in a solution of 10% formalin (Wako Pure Chemical
Industries, Ltd.): 25% glutaraldehyde (Nacalai Tesque, Inc., Kyoto, Japan) (9:1) at 4°C,
and thereafter embedded in paraffin. H&E stained specimens were prepared under routine
method.
8
F-Actin Staining of Ciona Eggs/Embryos
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For F-actin staining, dechorionated Ciona eggs/embryos were fixed with a 1:1 mixed solution of extraction buffer (2% Triton-X 100, 50 mM MgCl2, 10 mM KCl, 10 mM EGTA, 20% glycerol, 25 mM imidazole) and fixation buffer (0.25% glutaraldehyde (Nacalai Tesque), 3.7% formaldehyde, 100 mM HEPES (pH = 7.0), 50 mM EGTA, 10 mM MgSO4, and 525 mM sucrose) [21 (link)] for 15 min at room temperature. This was followed by continued fixation in fixation buffer for 2 h at room temperature. The fixed specimens were washed with PBST and treated with 10 unit/mL Alexa Fluor 488-labeled phalloidin (Molecular Probes, Eugene, OR, USA) for 30 min at room temperature. For clearing without dehydration, stained specimens were treated with 40% fructose (Fujifilm-Wako, Tokyo, Japan) for 30 min at room temperature and then mounted in SeeDB (80.2% wt/wt fructose, 0.5% α-thioglycerol) [22 (link)].
9
Transmission Electron Microscopy of Cells and EVs
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The ultrastructure of cells and EVs was observed by transmission electron microscopy as previously described8 (link) with some modifications. Briefly, sorted cells and EVs were fixed with 2.5% glutaraldehyde (Nacalai Tesque), 2% paraformaldehyde (Wako) in 0.1 M phosphate buffer (pH 7.4) at 4 °C overnight. Cells were then dehydrated and embedded in Epon 812 (TAAB Laboratories). Ultrathin sections were obtained from the Epon blocks and stained with uranyl acetate and lead citrate. For negative staining of EVs, a grid with a supporting film was placed on a drop of the EV suspension for 15 min and excess solution was removed with a filter paper. The grid was then placed in 2% uranium acetate aqueous solution for 2 min, followed by air-drying. Sections and stained EVs were observed under an electron microscope (H-7650, Hitachi).
10
Neonatal Brain Ultrastructural Analysis
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Transmission electronic microscopy was performed as previously described with minor modifications [17 (link)]. Briefly, the male pups in each group were analyzed at 24 h after birth. To induce hypothermia as anesthesia, they were immersed up to the neck in crushed ice and water and transcardially perfused with 2% paraformaldehyde and 2% glutaraldehyde (Nacalai Tesque) in 0.1 M PB (pH 7.2). The brains were dissected out, postfixed in the same fixative overnight at 4°C and sectioned at 1 mm using a brain matrices (Brain Science Idea. Co., Ltd., Osaka, Japan). Brain slices were post fixed with 1% OsO4, dehydrated with a graded series of ethanol, and embedded in Epon812 (Okenshoji, Tokyo, Japan). Semithin and ultrathin sections were cut with an ultramicrotome UC6 (Leica Microsystems, Vienna, Austria). Semithin sections were stained with tolidine blue and observed with an optical microscope (BX-51). Ultrathin sections were stained with uranyl acetate and lead citrate and examined with a transmission electron microscope HT7700 (Hitachi, Tokyo, Japan).
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