After 8, 12, 24, 36, 48, 60, 72,84, 96, 108, 120, and 132 h incubation on the plastic sheet, the suspension was removed and washed by 1 mL of 0.1M PBS. Then the samples were fixed for 30 min at 4°C with 4% glutaraldehyde (Sangon Biotech, Co., Ltd., Shanghai, China), and rinsed with 0.1M PBS to remove the glutaraldehyde, then stained with SYBR Green I (Sangon Biotech, Co., Ltd., Shanghai, China) in the dark for 30 min at room temperature. The excess stain was removed using PBS then air-dried. CLSM images were acquired using the confocal laser scanning machine (LSM710, Carl Zeiss AG, Germany). A 20× microscope objective was used, the SYBR Green I was excited at 488 nm and emitted at 525 ± 25 nm. For each sample, six separate sites were acquired randomly. The CLSM images were analyzed by the ISA-2 software (Ping Chen, Shanghai Ocean University, China) to determine biofilms structural parameters including biovolume and mean thickness (Tan et al., 2018 (link)).
Glutaraldehyde
Manufactured by Sangon
Sourced in China
Glutaraldehyde is a clear, colorless liquid used as a fixative and cross-linking agent in various laboratory applications. It is commonly employed in the preservation and preparation of biological samples for microscopic examination. Glutaraldehyde functions by reacting with amino groups in proteins, forming stable covalent bonds that help maintain the structural integrity of cells and tissues.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710, by Zeiss (6 mentions)
Sybr green 1, by Sangon (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Jem 1011 electron microscope, by JEOL (2 mentions)
Lead citrate, by Sangon (2 mentions)
Epon 812, by Sangon (2 mentions)
Formvar carbon coated nickel grids, by Sangon (2 mentions)
Uranyl acetate, by Sangon (2 mentions)
Progesterone, by Merck Group (1 mentions)
Pluronic f 127, by Merck Group (1 mentions)
Fluo 3 am, by Beyotime (1 mentions)
Bovine serum albumin (bsa), by Sangon (1 mentions)
Cysteine, by Solarbio (1 mentions)
Nocodazole, by Merck Group (1 mentions)
Higene, by Applygen (1 mentions)
Ex 527, by Merck Group (1 mentions)
Oligofectamine, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Zen 2011 program, by Zeiss (1 mentions)
15 protocols using glutaraldehyde
1
Biofilm Structural Characterization by CLSM
2
Protein Delivery and Imaging in Cells
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BioPORTER® QuikEase™ Protein Delivery Kit, pluronic F127, progesterone, IP3R1 antibody (rabbit), and fluorescein isothiocyanate-labeled peanut (Pisum sativum) agglutinin (FITC-PSA) were purchased from Sigma-Aldrich Chemical Co. (St Louis, MO, USA). Earle's balanced salt solution (EBSS), phosphate-buffered saline (PBS), and penicillin–streptomycin were obtained from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Sodium pyruvate and triethanolamine were purchased from Amresco, Inc. (Solon, OH, USA). Hoechst 33258 and polyvinylpyrrolidone-40 (PVP-40) were obtained from Shanghai Maokang Biotechnology Co., Ltd. (Shanghai, China). Bovine serum albumin (BSA) and glutaraldehyde were purchased from Sangon Biotech Co. (Shanghai, China). Cysteine and chlortetracycline (CTC) were obtained from Beijing Solarbio Science and Technology Co., Ltd. (Beijing, China). Rabbit IgG was purchased from Abcam Plc. (Cambridge, MA, USA). Fluo-3AM was obtained from Beyotime Biotechnology (Shanghai, China). The calcium ionophore A23187 was obtained from 4A Biotech Co., Ltd. IP3 was purchased from MedChem Express LLC (Monmouth Junction, NJ, USA). The FITC labeling kit was obtained from Elabscience Biotechnology Co. (Wuhan, China). All other chemicals were reagent grade.
3
Modulation of SIRT1 Deacetylase Activity
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Nocodazole, EX527, DMSO, C3742, and C646 were all obtained from Sigma, Lipofectamine 2000 was from Invitrogen, and Higene was from APPLYGEN. Glutaraldehyde was purchased from Sangon Biotech. The Flag-tagged SIRT1 WT or deacetylase-inactive mutant (HY) and SIRT1 siRNA retrovirus have been previously described [23 (link), 52 , 53 (link)]. SIRT1 and CHK2 siRNA retrovirus were purchased from GeneChem. The method of making special gene stable knockdown cells was as described previously [53 (link)]. In the knockdown experiments, cells were transfected using Oligofectamine (Invitrogen) according to the manufacturer’s protocol. Infected cells were identified by western blotting after 36 h of RNAi transfection. Stable clones of cells stably expressing Flag-SIRT1 WT, Flag-SIRT1 HY, or the empty vector Flag we used were selected by hydromycin (1000 mg/ml, Sigma). Site-directed mutagenesis of Flag-CHK2 was performed using QuikChange XL (Stratagene) and confirmed by sequencing.
4
Quantitative Analysis of Bacterial Biofilms
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After 12, 24, 36, 48, 60, and 72 h incubation, the strain VP-S36 biofilms formed on glass were washed by immersing in 1 mL of 0.1 M PBS. Then the biofilms were fixed for 30 min at 4°C in a solution containing 4% glutaraldehyde (Sangon Biotech Co., Ltd., Shanghai, China). After that, the glass was first gently rinsed three times with 1 mL of 0.1M PBS and stained with SYBR Green I (Sangon Biotech Co., Ltd., Shanghai, China) for 30 min in the dark at room temperature, after which the excess stain was removed and air dried. CLSM Images were acquired using the confocal laser scanning machine (LSM710, Carl Zeiss AG, Germany) using a 20× objective. SYBR Green I was excited using an argon laser at 488 nm and a 525 ± 25 nm band-pass filter was used to collect the emission. Then image stacking was acquired with a 1 μm thickness for each sample at six random fields of the slices. Image processing and analysis were performed using the Zen 2011 program (Carl Zeiss). The CLSM images were analyzed by the ISA-2 software (Professor Haluk Beyenal, Montana State University, United States) to determine biofilms structural parameters such as biovolume, mean thickness, porosity.
5
Chlorine-Induced Ultrastructural Changes in Salmonella
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In order to observe the morphology differences of the Salmonella cells with 0, 50, and 100 mg/L of chlorine, ultrastructural changes of bacterial cells were observed with a transmission electron microscope (TEM, Hitachi 7650, Ibaraki, Japan). The treated samples were removed from the water bath and placed into a sterile stomacher bag containing 25 mL BPW and then squeezed by hand to wash off attached bacteria. The wash solution for each treatment was collected into a 50 mL centrifuge tube and centrifuged at 6000× g for 10 min. The bacterial pellets were transferred into sterile 1.5 mL centrifuge tubes. Samples were fixed with 1 mL of 2.5% (v/v) glutaraldehyde (Sangon Biotech, Shanghai, China) and examined using a TEM as previously described [31 (link)].
6
Ultrastructural Changes of Salmonella
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In order to reconfirm the destruction of the Salmonella cells during scalding, ultrastructural changes of bacterial cells were observed with a transmission electron microscope (TEM, Hitachi 7650, Ibaraki, Japan). The treated samples were removed from the water bath and placed into a sterile stomacher bag containing 25 mL BPW and then squeezed by hand to wash off attached bacteria. The wash solution for each treatment was collected into a 50 mL centrifuge tube and centrifuged at 6000 × g for 10 min. The bacterial pellets were transferred into sterile 1.5 mL centrifuge tubes. Samples were fixed with 1 mL of 2.5% (v/v) glutaraldehyde (Sangon Biotech, Shanghai, China) and examined using a TEM as previously described [28 (link)].
7
SEM Analysis of Biofilm Structural Changes
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Scanning electron microscopy (SEM XL20, Philips, Amsterdam, The Netherlands) was used to investigate the structural modifications of biofilms after treatment with Qe, Ag NPs, or QA NPs. For biofilm formation, the overnight E. coli strain ECDCM1 was diluted to a ratio of 1:50 into fresh LB medium complemented with 0.5% milk solution. The cultures were added to 12-well plates (Costar; Corning, Steuben, NY, USA), when necessary, Qe, Ag NPs, or QA NPs were added for a final concentration of 5 µg/mL. Sterile 18 mm ×18 mm coverslips were placed in the wells and served as the attaching surface for the cells. The plates were incubated for 15 h at 37 °C. After incubation, the coverslips were taken out and washed three times with PBS (pH 7.4). The preparation of the samples for SEM was performed as follows(Sun et al., 2017 (link)) . The samples were fixed overnight with 2.5% glutaraldehyde (Sangon, Shanghai, China) at 4 °C and subsequently dehydrated using serial ethanol concentrations: 30, 50, 70, 80, 95 and 100%. Each ethanol treatment lasted for 20 min at 4 °C and, finally, the cover slips were freeze-dried overnight.
8
Analyzing Cell Migration in Co-culture
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The constructed control, sh LILRB1#1, sh LILRB1#2 HMC3 cells were sown in the lower chamber containing 600 µl of complete medium (DMEM and 20% FBS) and U87, U251 cells were put in the upper chamber with Matrigel (Corning Inc.) or without Matrigel containing serum-free DMEM. They were respectively co-cultured in 24 well culture plates with 8 μm Pore Polycarbonate Membrane (Corning Inc.) (2 × 104/well) at 37 ℃, 5% CO2 incubator. The cells in the upper chamber were co-cultured for 48 h before being fixed for 15 min with 4% glutaraldehyde (Sangon Biotech (Shanghai) Co.,Ltd.) and stained for 5 min with 1% crystal violet (Beyotime) at ambient temperature. The cells that had not migrated through the well were taken out using a cotton swab. Cells were photographed (magnification, ×100) and counted.
9
Ultrastructural Analysis of Epididymal Samples
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The segmented epididymal samples (caput, corpus, and cauda) were dissected and washed three times with PBS and fixed in 2.5% glutaraldehyde (0.2 M PBS, pH = 7.2; Sangon, Shanghai, China) overnight at 4 °C. The epididymal samples underwent dehydration using a series of alcohol concentrations: 30%, 50%, 70%, 80%, 95%, 100%, and two rounds of 100% acetone, each lasting 20 min for alcohol and 15 min for acetone. The treated samples were embedded in epoxypropane resin, sliced into 50 nm sections using an EM UC7 ultramicrotome (Leica, Wetzlar, Germany), stained with lead citrate and uranium, and observed using an HT7700 TEM (Hitachi, Tokyo, Japan).
10
Cross-linking Analysis of Bacterial Proteins
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Purified DrRuvC solution (0.1 mM) or DrYqgF (0.1 mM) was dialyzed against a buffer containing 20 mM HEPES (pH 8.0), 100 mM NaCl, and 10% glycerol, and it was treated with the indicated amounts of freshly diluted glutaraldehyde (0.01, 0.02, 0.04, 0.08, 0.16, and 0.32%) (Sangon, Shanghai) or bis-sulfo-succinimidyl suberate (BS3) (0.125, 0.25, 0. 5, 1, 2, and 4 mM) (Thermo Fisher Scientific, USA). After incubation at 25°C for 30 min, samples were treated with amine-containing quenching buffer (Tris [pH 8.0], with a final concentration to 100 mM) and then further incubated at 25°C for 10 min. Then, SDS-PAGE sample loading buffer was added. Samples were boiled for 10 min and analyzed by 12% (for DrRuvC) or 15% (for DrYqgF) SDS-polyacrylamide gel electrophoresis. The cross-linked and non-cross-linked bands were visualized via staining with Coomassie Brilliant Blue.
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