Rhodamine-labeled tubulin was prepared as previously described (Hyman, 1991 (link)). For the colocalization assay, taxol-stabilized microtubules composed of tubulin mixed with rhodamine-labeled tubulin (molar ratio 1:4) in PEMT were incubated with 0.5 μM ICR2-His6 for 15 min at 37°C and then crosslinked with 20 mM 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (Pierce Biotechnology) for 5 min at 37°C. The mixture was then centrifuged at 12,000 g for 5 min, and the pellet was resuspended in PEM buffer preheated to 37°C. ICR2-His6 was stained with an anti-His antibody (Sigma-Aldrich, H-1029, 1:5000) and a secondary antibody conjugated with fluorescein (Sigma-Aldrich, F0257, 1:5000). The solution was then centrifuged at 12,000 g for 5 min, and the pellet was resuspended with PEM buffer preheated to 37°C. An aliquot of 1 μl was put on a poly-L-lysine-coated glass slide (Sigma-Aldrich, P0425) and observed by confocal microscopy. For the in vitro bundling assay, the same taxol-stabilized rhodamine-labeled microtubules were incubated with 0.1, 0.5, 1 or 2 μM ICR2-His6 for 30 min at 37°C and then treated with 0.005% glutaraldehyde. A 1-μl aliquot of each sample was put on a poly-L-lysine-coated glass slide (Sigma-Aldrich, P0425) and observed by confocal microscopy.
Poly l lysine coated glass slide
Manufactured by Merck Group
Sourced in United States, United Kingdom
Poly-L-lysine coated glass slides are a type of laboratory equipment used for various cell culture and microscopy applications. The slides are coated with the positively charged amino acid polymer poly-L-lysine, which helps cells adhere to the surface more effectively. These slides are commonly used in techniques such as immunohistochemistry, in situ hybridization, and cell adhesion studies.
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Lab products found in correlation
Nocodazole, by Merck Group (2 mentions)
Paclitaxel, by Cambridge Bioscience (2 mentions)
Lr white resin, by Merck Group (2 mentions)
P0425, by Merck Group (1 mentions)
H1029, by Merck Group (1 mentions)
F0257, by Merck Group (1 mentions)
T7 rna polymerase, by Roche (1 mentions)
Paraplast, by Leica (1 mentions)
Digoxigenin rna labeling kit, by Roche (1 mentions)
Antifade solution, by Thermo Fisher Scientific (1 mentions)
Tcs sp2 aobs confocal microscope, by Leica (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Prolong glass antifade mountant, by Thermo Fisher Scientific (1 mentions)
Stellaris 8 inverted confocal microscope, by Leica (1 mentions)
Prolong gold antifade reagent, by Cell Signaling Technology (1 mentions)
U tv0.5xc 3, by Olympus (1 mentions)
Zen 2, by Zeiss (1 mentions)
Mitotracker deep red fm, by Thermo Fisher Scientific (1 mentions)
Calcofluor white m2r, by Merck Group (1 mentions)
28 protocols using poly l lysine coated glass slide
1
Colocalization and Bundling Analysis of Microtubules with ICR2-His6
2
In Situ Hybridization of Anther Transcripts
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Inflorescences were fixed in FAA buffer containing 3.7% (v/v) formaldehyde and vacuum-infiltrated for 15 min on ice. Samples were dehydrated in a graded ethanol series and stained with safranine in xylene/ethanol solutions. Samples were placed into a 60 °C oven for 1 wk and finally embedded in Paraplast (Leica, Germany). Transverse sections of 8 μm in thickness were transferred onto poly-L-lysine coated glass slides (Sigma-Aldrich, USA) for hybridization. RNA in situ hybridization was performed using a Digoxigenin RNA Labeling Kit (Roche, USA). A 497-bp SKS18 cDNA fragment and a 449-bp VTC1 cDNA were amplified and cloned into the pBluescriptSK vector. These plasmids were individually digested by BamHI or EcoRI and used as templates. Sense and antisense probes were transcribed using the above templates by the T3 or T7 RNA polymerase (Roche, USA), respectively. The hybridization for SKS18 transcripts in WT and tdf1 anthers was performed in a single batch, while that in ams was performed in another batch. The hybridization for VTC1 transcripts in WT and tdf1 anthers was performed in a single batch. Primer sequences are provided in Supplemental Data Set S1 .
3
Quantifying Spiroplasma Density via qPCR
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Spiroplasma density was quantified by qPCR using the dnaA Spiroplasma specific primers FqdnaA/RqdnaADoud for 35 cycles at 56 °C and normalized to the host β-tubulin gene. Primers and a detailed description used for the qPCR experiments are presented in Supplementary Table6 . qPCR data were analysed using a one-way ANOVA method, as described previously93 (link) using the XLSTAT program.
Gff specimens from the Seibersdorf laboratory colony were used for FISH. Teneral male and female flies were dissected in PBS 2–3 days after eclosion. Dissected tissues were dried on poly-L-lysine-coated glass slides (Sigma, UK) for 20 min at 65 °C and kept at 4 °C until further use. Tissue samples were fixed in freshly prepared 4% paraformaldehyde solution for 30 min at 4 °C. A detailed description of tissue processing and image capture is included in the Supplementary Information.
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Spiroplasma density was quantified by qPCR using the dnaA Spiroplasma specific primers FqdnaA/RqdnaADoud for 35 cycles at 56 °C and normalized to the host β-tubulin gene. Primers and a detailed description used for the qPCR experiments are presented in Supplementary Table
Gff specimens from the Seibersdorf laboratory colony were used for FISH. Teneral male and female flies were dissected in PBS 2–3 days after eclosion. Dissected tissues were dried on poly-L-lysine-coated glass slides (Sigma, UK) for 20 min at 65 °C and kept at 4 °C until further use. Tissue samples were fixed in freshly prepared 4% paraformaldehyde solution for 30 min at 4 °C. A detailed description of tissue processing and image capture is included in the Supplementary Information.
4
Quantifying Shrimp Hemocyte Apoptosis
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Shrimp hemocytes were collected and separated on poly-l -lysine-coated glass slides (Sigma), followed by standing for 10 min at 4°C. The hemocytes were fixed in 4% paraformaldehyde for 25 min at 4°C. The fixed hemocytes were washed with cold PBS and then subjected to the permeabilization with 0.2% Triton X-100 for 5 min. Next, the hemocytes were equilibrated in 100 µl of equilibration buffer at 4°C for 10 min. The equilibrated hemocytes were counterstained with PI after incubation with rTdT mix in a humid environment for 1 h. Subsequently, 2× SSC (1× SSC is 0.15 M NaCl and 0.015 M sodium citrate) was added to the slide to stop the reaction. The slide was covered with antifade solution (Invitrogen) to prevent signal quenching.
5
Confocal Microscopy Imaging Protocol
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Stained cells were allowed to adhere to poly-L-lysine–coated glass slides (Sigma-Aldrich) and mounted with antibleach reagent (Vector Laboratories). Samples were analyzed using a 63×/1.4 HCX PL APO CS oil objective on a TCS SP2 AOBS confocal microscope (Leica). Images were acquired using LCS 2.61 (Leica) and processed using ImageJ (National Institutes of Health).
6
Optimizing Metaphase Spreads for Analysis
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Cell lines were optimized to generate metaphase spreads. Briefly, cells at near confluence in a T75 flask were incubated between 4 and 16 hr in the presence of 10–100 nm paclitaxel (Cambridge BioScience) with or without 50–100 ng/ml nocodazole (Sigma-Aldrich). Along with the media, cells dissociated with accutase were centrifuged, washed in PBS, and resuspended in 10 ml potassium chloride (KCl) 0.56%, with sodium citrate dihydrate (0.9%) if required, for 20 min. After further centrifugation, cells were resuspended in methanol:acetic acid 3:1 and dropped onto humidified slides.
For all other fixed cell experiments described below, cells were seeded overnight onto glass cover-slips or poly-L-lysine coated glass slides (Sigma-Aldrich). Cells were fixed with 4% paraformaldehyde (PFA – 10 min) and permeabilized with 0.5% Triton X-100 (15 min) with thorough PBS washes in-between. Where cells were dried (see FISH methods), this only occurred following PFA fixation in order to preserve 3D structures and minimize cell and nuclear flattening.
For all other fixed cell experiments described below, cells were seeded overnight onto glass cover-slips or poly-L-lysine coated glass slides (Sigma-Aldrich). Cells were fixed with 4% paraformaldehyde (PFA – 10 min) and permeabilized with 0.5% Triton X-100 (15 min) with thorough PBS washes in-between. Where cells were dried (see FISH methods), this only occurred following PFA fixation in order to preserve 3D structures and minimize cell and nuclear flattening.
7
Visualizing CD8+ T-cell-Platelet Interactions
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After overnight coculture, CD8+ T-cell–platelets were prepared for confocal imaging. Cells were fixed with 4% paraformaldehyde for 10 minutes in 5% CO2 at 37°C, washed, and blocked with 2% bovine serum albumin (Thermo Fisher Scientific) for 1 hour. Cells were stained with purified antihuman CD42b monoclonal antibody (BioLegend), followed by goat antimouse immunoglobulin G (H + L)–Alexa Fluor Plus 555 (Thermo Fisher Scientific) before staining for Alexa Fluor 594–conjugated antihuman CD8a (BioLegend). Nucleic acid staining of cells was performed using Hoechst-33342 (Thermo Fisher Scientific), and cells were mounted in ProLong glass antifade mountant (Thermo Fisher Scientific) on poly-L -lysine–coated glass slides (Sigma-Aldrich, United Kingdom). Cells were analyzed using a Stellaris 8 inverted confocal microscope (Leica, United Kingdom).
8
NFκB Nuclear Translocation Assay
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A549 cells were seeded on poly-L-lysine-coated glass slides (Catalog#: p0425; Sigma-Aldrich) and cultured in a 35 mm Petri dish with 3 mL of media at 37°C in a humidified atmosphere of 5% CO2. After 24 h, cells were pre-incubated with or without 0.3 nM MC4 or FK866 for 2 h. Then, cell layers were treated with TNFα (10 ng/mL), TNFα plus 0.3 nM of MC4, or TNFα plus 0.3 nM of FK866 for an additional 20 min. Cell layers were washed three times with 5 mL PBS for 5 min each and fixed in 4% paraformaldehyde for 15 min at RT. Afterward, the slides were rinsed three times in 2 mL of PBS for 5 min each, followed by 60 min in blocking buffer (PBS/5% normal goat serum; Catalog#: 5245; Cell Signaling Technology). The slides were incubated overnight at 4°C with NFκB antibody (1:500) in antibody dilution buffer (PBS/1% BSA/0.3% Triton™ X-100) followed by three washes with PBS for 5 min each. Incubation of the slides in diluted anti-rabbit IgG (H+L), F(ab′)2 fragment (Alexa Fluor® 488 Conjugate) with 1:50 antibody dilution buffer for 1 h at RT in the dark. The slides were then washed (3×) with PBS, prior to attaching coverslips with Prolong Gold Antifade Reagent (Catalog#: 9071; Cell Signaling Technology) with 4,6-diamidino-2-phenylindole (DAPI). The slides were cured for 24 h at RT prior to microscopy (Olympus, U-TV0.5xC-3, Center Valley, PA, USA).
9
Live Cell Confocal Imaging of Yeast
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The acquisition of confocal images of live yeast cells was performed using a Zeiss LSM800 confocal microscope with a 63× 1.4 oil immersion objective and ZEN 2.3 software (Zeiss). Yeast that expressed selected IMS proteins that were tagged with tFT were grown in minimal synthetic media with glycerol as a main carbon source at 24 °C. Prior to imaging, growth media were supplemented with MitoTracker Deep Red FM (200 nM, Thermo Scientific) and Calcofluor White M2R (5 μg/ml, Sigma), followed by 10 min of incubation. For imaging, the samples were placed on poly-l -lysine-coated glass slides (Sigma). The acquisition settings were adjusted for each experimental condition for maximum intensity projections. Sixteen-bit images with a resolution of 358 × 358 pixels were acquired, exported to Adobe Photoshop software with adjustments of contrast and brightness, and assembled in Adobe Illustrator software.
10
Visualizing Nucleoid Structure in Topoisomerase Mutants
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Cultures of strains PZntopA and ΔtopAPZntopA were grown at mid-log growth phase in media with different amounts of ZnSO4. Samples (from 5 × 107 to 2 × 108 cells) were collected, washed in 10 mM phosphate buffer (pH 7.2), and fixed in 2% of paraformaldehyde for 48 h. Fixed cells were washed, suspended in buffered salt solution (137 mM NaCl, 5.4 mM KCl, 10 mM Tris–HCl, and pH 7.6) and incubated with 5 μM Sytox™ Orange Nucleic Acid Stain (Invitrogen) for 5 min at room temperature. ProLong™ Gold Antifade Mountant (Invitrogen) was added to fixed cells, and the mixture was transferred onto poly-L-lysine coated glass slides (Sigma-Aldrich). Slides were observed using a confocal microscope STELLARIS 8–FALCON/STED (Leica Microsystems) with a HC PL APO 100×/1.40 NA × OIL immersion objective. Super resolution images were acquired by Stimulated Emission Depletion (STED) microscopy using 660nm depletion laser. Image J software was used for image analysis. Average Sytox fluorescence intensity was measured from the nucleoids, defined as the region of each cell with intensity values between 20 and 225. Raw integrated density measurements divided by area (Mean Gray Values) were obtained for 1,307–4,562 nucleoids. GraphPad Prism 9.1 was used to represent the average intensities vs. Sc density (σ) and to perform a simple linear regression.
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