For confocal microscopy, macrophages were seeded in black poly-d-lysine coated glass 96-well plates (MatTek Corporation, Ashland, MA, USA). To stain lysosomes, macrophages were incubated with 75 nM Lysotracker Red or Deep Red (Thermo Fisher Scientific) at 37°C/5%CO2 for 1 h before fixation. Cells were fixed for 1 h in 1% EM-grade formaldehyde, followed by quenching with PBS/1.5 mg/ml glycine for 10 min and blocking in 5% human serum for 45 min, all at room temperature. For immunostaining, cells were permeabilized for 10 minutes with 0.1% Triton X-100 before blocking and subsequently stained with primary and secondary antibodies for 30 minutes each in the dark at room temperature. Finally, cells were stained with phalloidin-Alexa488 (Thermo Fisher Scientific) and/or LipidTOX Green (Thermo Fisher Scientific) for 30 min according to the manufacturers’ instructions, and/or 50 μg/ml Filipin complex from Streptomyces filipinensis (Sigma-Aldrich) for 2 h at room temperature in the dark. Lysotracker and filipin pictures were taken using a SP8WLL confocal microscope (Leica, Amsterdam, The Netherlands). Galectin-3 and NDP52 colocalization was visualized using a Dragonfly spinning-disk confocal microscope (Andor Technologies, Belfast, UK) equipped with 405, 488, 561 and 640nm lasers and a Zyla 4.2 sCMOS camera.
Phalloidin alexa488
Manufactured by Thermo Fisher Scientific
Sourced in United States
Phalloidin-Alexa488 is a fluorescent labeling reagent used for the detection and visualization of F-actin in cells and tissues. It binds specifically to F-actin and can be detected using fluorescence microscopy.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Ix81 confocal microscope, by Olympus (2 mentions)
Triton x 100, by Reanal (2 mentions)
Draq5, by BioLegend (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Cycloheximide (chx), by Merck Group (2 mentions)
Lsm 510, by Zeiss (2 mentions)
Lipidtox green, by Thermo Fisher Scientific (1 mentions)
Filipin complex from streptomyces filipinensis, by Merck Group (1 mentions)
Sp8 wll confocal microscope, by Leica (1 mentions)
Bodipy 493 503, by Thermo Fisher Scientific (1 mentions)
Mtc02, by Abcam (1 mentions)
Fv1000, by Olympus (1 mentions)
Perm wash buffer, by BD (1 mentions)
Spectramax m5e, by Molecular Devices (1 mentions)
Bz x710, by Keyence (1 mentions)
Lab tek, by Thermo Fisher Scientific (1 mentions)
35 protocols using phalloidin alexa488
1
Confocal Microscopy of Macrophage Lysosomes
2
Multiparametric Imaging of LNCaP-C4-2 Cells
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LNCaP-C4-2 cells were grown in 22 mm glass coverslips to 70% confluency. For imaging, coverslips were fixed with 4% Paraformaldehyde and permeabilized with perm/wash buffer from BD Biosciences (San Jose, CA, USA). Hybridization with primary antibodies was done using perm/wash solution in humidified chambers. For mitochondria staining we used MTC02 (Abcam 3298) at 1:500 dilution overnight, followed by a secondary TRITC-conjugated antibody at 1:1000 for 1 h. BODIPY™ 493/503 (Thermo Fisher, Waltham, MA, USA) was used at 1:1000 dilution for 10 min to stain lipid droplets. Phalloidin-Alexa 488 (Thermo Fisher, Waltham, MA, USA) was used at 1:1000 dilution to demarcate the cell perimeter and identify individual cells. DAPI was used to stain the nucleus. Images were taken with an Olympus FV1000 laser scanning confocal via a 100× UPlanSApo oil objective in the Advanced Light Microscopy Core at University of Colorado Denver, Anschutz Medical Campus (Aurora, CO, USA). Analysis was done with free software FIJI (https://imagej.nih.gov/ij/ ), using the ROI feature to identify individual cells and measuring the mitochondria (red) and lipid (green) channels. A minimum of 20 measurements were done per condition.
3
Immunohistochemistry for GPR18 Receptor
4
High-Content Analysis of CDT Toxin Effects
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Vero cells were plated in 96-well Cellbind plates at a density of 8000 cells/well (~90% confluent). The next day, compound dilutions and CDT binary toxins (2 μg/mL each of A and trypsin-activated B—a kind gift from Merck) in serum-free DMEM were added to the plate. After 24 h, wells were washed with PBS, and the cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.25% TX100 for 5 min, then F-actin was stained with Phalloidin Alexa488 (Thermo) for 2 h before washing and reading Alexa488 fluorescence on a Molecular Devices Spectramax M5e (bottom read, well scan, 9 points/well).
For photomicroscopy, the cells were stained with 1 μM Hoechst for 30 min, then combined photos were taken for each compound at a concentration corresponding to maximum protection from TcdB using a 10× objective and appropriate filter sets for Hoechst and Alexa488 fluorescence (Zeiss).
For photomicroscopy, the cells were stained with 1 μM Hoechst for 30 min, then combined photos were taken for each compound at a concentration corresponding to maximum protection from TcdB using a 10× objective and appropriate filter sets for Hoechst and Alexa488 fluorescence (Zeiss).
5
Quantifying Cell Size Changes
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Cell size analysis was assessed by fluorescence staining with phalloidin-Alexa 488 (Thermo Fisher Scientific, cat#: A12379, Waltham, MA, USA). NRCMs were seeded onto 24 well plates and cultured overnight. After a 2 day period of differentiation, Dox (3 mmol/L) and HSA-Trx (1–10 mmol/L) in D-MEM were added and incubated at 37 °C for 12 h. After washing with PBS, 4% PFA in PBS was added and incubated for 10 min at room temperature. Permeabilization was achieved by adding 0.5% TrironX-100 in a Tris-buffered saline solution (TBS). After washing with 0.1% TritonX-100 in TBS and blocking with 0.1% TrironX-100, 1% BSA in TBS, the antibody reaction was performed by phalloidin-Alexa 488 in blocking solution. The cells were observed by a fluorescence microscope (BZ-X710; Keyence, Osaka, Japan) and analyzed using the Image J software (National Institutes of Health and LOCI, University of Wisconsin, Madison, WI, USA).
6
Actin Cytoskeleton Visualization in AOXlar7y Cells
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A total of 10,000 AOXlar7y cells were plated on chamber slides (LabTek; Thermo Fisher, Dreieich, Germany) and cultured as described above. After the cultivation period, the cells were fixed in 3% paraformaldehyde solution at room temperature for 10 min, permeabilized with 0.1% saponin for 10 min, and washed thrice with PBS. To image the actin filaments (cytoskeleton component) of the cells, Phalloidin Alexa 488 (Thermo Fisher) was added (5 µL in 200 µL PBS) at room temperature for 20 min. Subsequently, the cells were washed thrice with PBS, followed by DAPI staining (Roth, Karlsruhe, Germany; diluted 1:1000 in PBS) for five minutes to visualize the nuclei. The slides were finally rinsed in PBS and aqua dest. and embedded in DAKO fluorescence mounting medium (Roth). Images were taken with a BZ-9000 microscope (Keyence).
7
Immunofluorescent Staining of CD4+ T Cells
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CD4+ T cells cultured in 16-well Lab-Tek chamber slides (Thermofisher) were fixed with 4% paraformaldehyde for 20 min. Fixed cells were washed twice with PBS, and permeabilized with Perm buffer (0.1% Triton X-100 in PBS). Following fixation, cells were stained with anti-Foxo1 (CST) in Perm buffer for 30 min, washed twice, and then stained with anti-rabbit Alexa647 secondary antibody (Thermofisher) for 1 h. Cells were washed again with Perm buffer, and stained with Phalloidin Alexa488 (Thermofisher) for 1 h. Lastly, cells were washed a final time with PBS, and then mounted in DAPI Diamond Antifade (Thermofisher).
8
Graphene Oxide-PEI for Osteogenic Differentiation
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The following materials were purchased from various companies. Graphene oxide (Graphene super market, Reading, MA, USA), N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC, Sigma-Aldrich, St. Louis, MO, USA), Polyethyleneimine (PEI, ~Mw 25k, Sigma-Aldrich, St. Louis, MO, USA), dialysis membrane (pore size 30k), simvastatin (Sigma-Aldrich, St. Louis, MO, USA), and fetal bovine serum (FBS, Hyclone, Waltham, MA, USA). penicillin/streptomycin (P/S, Gibco, Waltham, MA, USA), Dulbecco’s modified Eagle medium (DMEM, Welgen, Gyeongsan-si, Korea), β-glycerophosphate (Sigma-Aldrich, St. Louis, MO, USA), Dexamethasone (Sigma-Aldrich, St. Louis, MO, USA), l -ascorbic acid (Sigma-Aldrich, St. Louis, MO, USA), MTT assay (Thermo Fisher Scientific, Waltham, MA, USA), paraformaldehyde (PFA, Sigma-Aldrich, St. Louis, MO, USA), Phalloidin Alexa 488 (Thermo Fisher Scientific, Waltham, MA, USA), ALP detection kit (Biovision, Milpitas, CA, USA), Bradford assay buffer (Thermo Fisher Scientific, Waltham, MA, USA), Alizarin Red S (ARS, Sigma-Aldrich, St. Louis, MO, USA), cDNA Synthesis Kit (Nanohelix, Daejeon, Korea), and primers including RUNX2, OPN, and OCN (Bioneer, Daejeon, Korea).
9
Sponge-Algae Symbiosis Imaging
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Sponges were grown on 35 mm glass bottom dishes (MatTek Life Sciences) and either infected with live sponge-derived algae or left untreated. Fixation and imaging were conducted as described in Hall et al. (2021) (link). Briefly, sponges were fixed in 4% paraformaldehyde and 1/4 Holtfreter’s Solution overnight at 4 °C. After washing and permeabilization, tissue was stained with Hoescht 33342 (1:200 dilution, Thermo Fisher Scientific, Waltham, MA) and Phalloidin Alexa 488 (1:40 dilution, Thermo Fisher Scientific, Waltham, MA) and imaged using an Olympus FV1200 laser scanning microscope using FluoView software.
Electron microscopy was performed as described in Hall et al. (2021) (link). Briefly, sponge samples infected with live algae were fixed in 2.5% glutaraldehyde, washed in 0.2 M cacodylate buffer (pH 7.4), and postfixed with 1% OsO4 and 1% uranyl acetate. Samples were dehydrated, infiltrated in propylene oxide, and embedded in Embed 812 plastic resin. Ultrathin sections were stained with uranyl acetate and lead citrate. Micrographs were taken using a JEOL 1010 transmission electron microscope at the University of Richmond with an Advanced Microscopy Techniques XR-100 Digital CCD system.
Electron microscopy was performed as described in Hall et al. (2021) (link). Briefly, sponge samples infected with live algae were fixed in 2.5% glutaraldehyde, washed in 0.2 M cacodylate buffer (pH 7.4), and postfixed with 1% OsO4 and 1% uranyl acetate. Samples were dehydrated, infiltrated in propylene oxide, and embedded in Embed 812 plastic resin. Ultrathin sections were stained with uranyl acetate and lead citrate. Micrographs were taken using a JEOL 1010 transmission electron microscope at the University of Richmond with an Advanced Microscopy Techniques XR-100 Digital CCD system.
10
Immunostaining of Hydrogel Samples
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Samples were washed with pre-warmed DPBS and fixed with 4% paraformaldehyde (#P6148, Sigma-Aldrich, St. Louis, MO, USA) in DPBS (#14040, ThermoFisher Scientific, Waltham, MA, USA) for 1 h at RT. After fixation, samples were washed three times with DPBS for 10 min. Hydrogel samples were incubated in 10% (v/v) goat serum (#G9023)/0.1% (v/v) Triton X-100 (#T8787)/PBS, and TUBB3 antibody (1:200, rabbit polyclonal, #T2200, all Sigma-Aldrich, St. Louis, MO, USA), at 4 °C overnight. Specimens were washed three times for 2 h each with DPBS and subsequently incubated with anti-rabbit-Alexa546 (1:500, #A11010, Invitrogen), Phalloidin-Alexa488 (1:70, #A12379, ThermoFisher), and Hoechst (1%, bis-benzimide H33258, #B1155, Sigma) in 2% goat serum/PBS over night at 4 °C. Subsequently, samples were washed three times for 2 h each in PBS at RT and directly imaged by a confocal laser scanning microscope (CLSM, TCS SP8, Leica Microsystems, Wetzlar, Germany) using the objectives HC PL FLUOTAR 5x/0.15 DRY, HC PL APO CS2 10x/0.40 DRY, and HC PL APO CS2 20x/0.75 DRY. Maximum intensity projections of recorded z-stacks were constructed using Fiji Image J 1.52p [34 (link)]. ADA-GEL hydrogel served as control to LAM-supplemented ADA-GEL hydrogels.
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