For non-specific staining of cell membrane and contents, 0.05% (w/v) Toluidine Blue O (T3260, Sigma-Aldrich) in 0.1 M phosphate buffer pH 6.8 was added to the fruit tissue in the tube. After staining for 5 min, the stained samples were mounted onto poly-L-lysine coated slides (Polysine, J2800AMNZ, Thermo-Scientific). For starch staining, the fruit tissue was dispersed in distilled water and placed on a polysine coated slide, then one drop of Gram’s iodine solution (90107, Sigma-Aldrich) was added and mixed directly on the slide. For cellulose staining, 0.1% (w/v) Calcofluor White stain [Fluorescent Brightener 28 (319945), Sigma-Aldrich] was added to fruit tissue in the tube. One drop of stained sample was placed on a polysine coated slide, then made alkaline with one drop of 10% (v/v) NaOH. The sample were observed using an inverted light microscope for Toluidine Blue O and iodine staining, and UV fluorescence microscope for Calcofluor White staining (Olympus, model BH2, Japan). Images were captured using a digital camera (Sony, model sCMEX-3). All staining was done at room temperature.
J2800amnz
Manufactured by Thermo Fisher Scientific
Sourced in United States
The J2800AMNZ is a high-speed centrifuge designed for laboratory applications. It features a maximum speed of 30,000 rpm and a maximum RCF of 50,000 x g. The centrifuge can accommodate a variety of rotor types and sample volumes to meet the diverse needs of researchers and scientists.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710 inverted microscope, by Zeiss (2 mentions)
Fluorescent brightener 28, by Merck Group (1 mentions)
Gram s iodine solution, by Merck Group (1 mentions)
Toluidine blue o, by Merck Group (1 mentions)
Ab22758, by Abcam (1 mentions)
Ab476, by Abcam (1 mentions)
Cytospin 4 centrifuge, by Thermo Fisher Scientific (1 mentions)
Axio imager z1 apotome microscope, by Zeiss (1 mentions)
Prolong gold, by Thermo Fisher Scientific (1 mentions)
A11008, by Thermo Fisher Scientific (1 mentions)
A7906, by Merck Group (1 mentions)
P6148, by Merck Group (1 mentions)
T8787, by Merck Group (1 mentions)
Nestin, by Merck Group (1 mentions)
Lcs sp5, by Leica camera (1 mentions)
Axioimager z2 epifluorescence microscope, by Zeiss (1 mentions)
Poly l lysine slide, by Thermo Fisher Scientific (1 mentions)
Vectashield h 1200, by Vector Laboratories (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
7 protocols using j2800amnz
1
Multi-staining Fruit Tissue Protocol
2
Immunofluorescence Staining of BLM and Nucleolin
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Cells were deposited on poly-lysine coated slides (J2800AMNZ, Thermo Scientific) using a Cytospin 4 centrifuge (Thermo Scientific) at 600 rpm for 10 minutes. Soluble cell fraction was pre-extracted by incubation with cold cytoskeleton buffer (CSK: 10 mM PIPES, pH 7, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 0.7% Triton X-100) (2 x 3 minutes), fixed with 4% PFA-PBS, and saturated with 3% BSA-PBS for 1h at RT. Anti-BLM antibodies (ab476, Abcam; sc-365753, Santa Cruz) were diluted at 1:200 and anti-nucleolin (ab22758, Abcam) was diluted at 1:1000 in saturation buffer and incubated on slides in a humid chamber for 90 minutes. Slides were washed 3 x 5 minutes with PBS-0.01% Tween, incubated protected from light in a humid chamber with secondary antibody (A11008, Invitrogen) 1:500 for 45 minutes at RT. Washed again 3 x 5 minutes with PBS-0.01% Tween, incubated with DAPI (20 μg/ml) in H2O for 5 minutes and washed 3 times with H2O. Slides were air dried and mounted with Prolong Gold (P36930, Invitrogen) and let to dry overnight. Image acquisition was performed with a ZEISS Axio Imager Z1 Apotome microscope and analysis was done with Omero server.
3
Immunofluorescence Staining and Microscopy
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Cells were seeded on 13 mm circular coverslips and placed in 24-well plates. After exposure to different experimental conditions, fixation was performed with 4% (v/v) paraformaldehyde (P6148, Merck, Darmstadt, Germany) for 10 min at room temperature (RT), permeabilization with 0.1% (v/v) Triton-X100 (T8787, Merck, Darmstadt, Germany) for 10 min and blocking with 10% bovine serum albumin (A7906, Merck, Darmstadt, Germany) containing 0.5% (v/v) Triton-X100 for 30 min at RT. Primary antibodies were incubated for 1 h at RT and secondary antibodies for 30 min at 4 °C in darkness. Finally, all coverslips were mounted onto slides (J2800AMNZ, Thermo scientific, Waltham, MA, USA), including DAPI (4′,6-diamidino-2-phenylindole) for nuclear counterstain. Images were acquired on a Confocal Zeiss LSM 710 inverted microscope with a 63× immersion objective. In a different manner, BG4 immunofluorescence was conducted as previously described [11 (link)]. BG4 mean nuclear fluorescence intensity was quantified using Fiji software (N > 250). Antibodies used are listed in Supplementary Table S1 .
4
Immunocytochemical Analysis of Neurospheres
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Cells grown as neurospheres were dropped on poly d-lysine-coated slides (Microscope slide Polysine adhesion Thermo Scientific J2800AMNZ) and fixed in PBS-PFA 4%. Next, cells were permeabilized with 1× PBS containing 0.5% Triton X-100. SOX2 and NESTIN (Millipore) were used at a dilution of 1:100, and nuclear counterstaining with DAPI was performed after removal of excess secondary antibody. Immunostaining was visualized with Confocal Leica LCS SP5. At least 250 cells in 10 independent fields of view per condition and replicate were analyzed in 3 independent experiments.
5
Visualizing Toxoplasma Gliding Trails
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107 freshly egressed parasites were resuspended in 300 μl of motility buffer (Ringer's solution: 155 mM NaCl, 3 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 3 mM NaH2PO4, 10 mM Hepes, 10 mM glucose). Hundred microliters were deposited on poly-L-lysine coated microscope slides (J2800AMNZ, Thermo Scientific), in a well delineated with a hydrophobic pen (PAP Pen, Kisker Biotech). Parasites were left to glide for 15 min in an incubator at 37 °C, then the suspension was carefully removed and parasites were fixed with 4% (w/v) PFA in PBS. Immunostaining was performed with an anti-SAG1 antibody (81 (link)) as described above, but without permeabilization. Alternatively, trails were stained with the DiI perchlorate lipophilic membrane stain (D282, Invitrogen). The gliding assay was performed as described above, with the modification that DiI was added at a final concentration of 10 μM directly in the Ringer’s solution for the duration of the gliding assay (15 min), prior to fixation with 4% (w/v) PFA in PBS and further processing for microscopic imaging.
Trail deposition images were acquired with a 63× objective on a Zeiss AXIO Imager Z2 epifluorescence microscope and processed with ImageJ v. 1.53f51, using the NeuronJ plugin as described previously (38 (link)).
Trail deposition images were acquired with a 63× objective on a Zeiss AXIO Imager Z2 epifluorescence microscope and processed with ImageJ v. 1.53f51, using the NeuronJ plugin as described previously (38 (link)).
6
Isolation and Visualization of Extracellular Vesicles
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Urine sample from a healthy donor (100 mL) was centrifuged in 3000g and next supernatant was ultracentrifuged in 150 000g for 1 h at 4°C. EVs pellet was resuspended in 60 μL of PBS and 20 μL of EVs solution was placed on 1 × 1 cm poly-l-lysine slide (Cat. number J2800 AMNZ, Thermo Fisher Scientific, Waltham, Massachusetts, USA) and incubated for 1 h in humid chamber at RT. After incubation the slide was washed twice in PBS and fixed in 3.7% glutaraldehyde in PBS for 30 min followed by salt removal stage. The slide with EVs was washed with two aqueous PBS dilutions, 50% PBS, 25% PBS, and deionized water, each for 1 minute. Next, the dehydration was applied by immersing sample for 30 seconds in ethyl alcohol solutions as follows: 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and absolute ethanol. Afterwards, sample was dried for 24 h under cover at RT [23 (link)].
The Environmental Scanning Electron Microscopy (ESEM) measurements were performed using SEM Quanta 3D FEG microscope by FEI Company (USA) operated at Institute of Physics Jagiellonian University, Kraków, Poland. The ESEM images were collected by GSED detector using electrons of 5 keV energy. During measurements the specimen was kept at 100 Pa of water vapor at RT.
The Environmental Scanning Electron Microscopy (ESEM) measurements were performed using SEM Quanta 3D FEG microscope by FEI Company (USA) operated at Institute of Physics Jagiellonian University, Kraków, Poland. The ESEM images were collected by GSED detector using electrons of 5 keV energy. During measurements the specimen was kept at 100 Pa of water vapor at RT.
7
Immunofluorescence Staining of G4 Structures
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Cells under different experimental conditions were seeded into 13-mm circular coverslips and placed in 24-well plates. Fixation was performed with 4% (v/v) paraformaldehyde (P6148, Sigma Aldrich) for 10 min at room temperature (RT), permeabilization with 0.1% (v/v) Triton-X100 (T8787, Sigma Aldrich) for 10 min and blocking with 10% bovine serum albumin (A7906, Sigma Aldrich), 0.5% (v/v) Triton-X100 for 30 min at RT. Primary antibodies were subsequently incubated for 1 h at RT and secondary antibodies for 30 min at 4 C in darkness. All coverslips were mounted onto poly-L-lysine slides (J2800AMNZ, Thermo scientific) with Vectashield (H-1200, Vector) including DAPI (4',6-diamidino-2-phenylindole) for nuclear counterstain. Images were acquired through a Confocal Zeiss LSM 710 inverted microscope with a 63x immersion objective. Antibodies used are listed in Supplementary files (Table S1A). In particular, the G4 selective antibody BG4 was expressed and used for immunofluorescence according to standard method (Biffi et al., 2013) (link).
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