Aortic SMC were fixed at 24 hrs after plating by immersion in 2% paraformaldehyde in Dulbecco’s phosphate buffered saline (DPBS). Cells were then washed in a glycine buffer and incubated overnight at 4 °C with primary antibodies against integrin α2 (Abcam, San Francisco, CA, USA), integrin α5 (Milipore Sigma, Burlington, MA, USA), and smooth muscle α-actin (SMα-actin) (Millipore Sigma, Burlington, MA, USA) diluted in a sodium citrate buffer containing BSA and Triton X [24 (link)]. After washing, cells were incubated with Alexa 568 secondary antibody (Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature, washed again and immediately imaged in DPBS. A similar procedure with overnight incubation was followed for the primary antibody against integrin β1 or β3 both pre-conjugated with Alexa 488 (BioLegends, San Diego, CA, USA). For smooth muscle γ-actin (SMγ-actin, Actg2) staining, cells were first fixed with 1% paraformaldehyde in DPBS followed by permeabilization with cold methanol [25 (link)]. Staining was performed as described by using an SMγ-actin primary antibody [26 (link),27 (link)] followed by Alexa 488 secondary antibody (Jackson Immuno Research, West Grove, PA, USA).
Alexa 488
Manufactured by BioLegend
Sourced in United States
Alexa 488 is a fluorescent dye that can be used to label and detect proteins, cells, or other biological molecules. It has an excitation maximum at 495 nm and an emission maximum at 519 nm, which makes it compatible with common fluorescence detection systems. The dye is stable, water-soluble, and can be used in a variety of applications, including flow cytometry, fluorescence microscopy, and immunoassays.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd44 or anti cd69 conjugated to alexa fluor 647, by BioLegend (2 mentions)
Cd45ro conjugated to pe cy5, by BD (2 mentions)
Primary anti cd4 clone rpa t4, by BioLegend (2 mentions)
Biotin xx tyramide superboost kit, by Thermo Fisher Scientific (2 mentions)
Integrin α5, by Merck Group (1 mentions)
Sm α actin, by Merck Group (1 mentions)
Alexa 488 secondary antibody, by Jackson ImmunoResearch (1 mentions)
Integrin α2, by Abcam (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Foxp3 apc, by Thermo Fisher Scientific (1 mentions)
Ifn γ pe, by BD (1 mentions)
Cd8 apc, by BioLegend (1 mentions)
Tnf α fitc, by BD (1 mentions)
Leukocyte activation cocktail, by BD (1 mentions)
Cd4 percp cy5, by BD (1 mentions)
Cd25 bv605, by Thermo Fisher Scientific (1 mentions)
Il4 alexafluor 488, by BioLegend (1 mentions)
Cd69 apc cy7, by BD (1 mentions)
Facsaria 3 flow cytometer, by BD (1 mentions)
Facscalibur, by BD (1 mentions)
13 protocols using alexa 488
1
Immunofluorescence Staining of Aortic SMCs
2
Multiparameter Flow Cytometry of KEAP1-Edited T Cells
3
Multiparameter Flow Cytometry Analysis
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Cell surface molecules were stained in PBS containing 1.5% FBS for 30 min at 4°C. Subsequently, cells were fixed with fixation buffer (eBioscience) for 30 min at 4°C and washed with permeabilization buffer (eBioscience). For Foxp3 staining, a Foxp3 staining kit (eBioscience) was used according to the manufacturer’s protocol. Intracellular cytokines and transcription factor were stained in the permeabilization buffer. The following antibodies were used: Alexa488 or PerCP-Cyanine5.5-conjugated anti-IFNγ (XMG1.2, Biolegend), Pacific Blue-conjuated anti-Foxp3 (MF–14, Biolegend), FITC-conjugated anti-TCR Vα2 (B20.1, Biolegend), PE-conjugated anti-IL–17 (TC11-18H10.1, Biolegend), PE-Cyanine7-conjugated anti-CD44 (IM7, Biolegend), PerCP-Cyanine5.5 or APC-Cyanine7-conjugated anti-CD4 (GK1.5, Biolegend), Alexa647-conjugated anti-IL–4 (11B11, Biolegend) and APC-conjugated anti-IL–5 (TRFIC5, Biolegend). Cells were analyzed with FACSCalibur, FACSVerse or FACSAria III flow cytometer (BD BioScience). Obtained data were analyzed with FlowJo software.
4
Live Imaging of Transgenic Parasites
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Live imaging of transgenic parasites expressing GFP-tagged DHHC2 was done by collecting tail blood samples from infected mice, mosquito blood meals at 16 hours p.i., as well as dissected mosquito midguts, midgut sporozoites and salivary gland sporozoites and staining with 1 μg/mL of Hoechst 33342/PBS. Red blood cell membranes were stained with anti-mouse TER-119 antibody conjugated with Alexa-488 (BioLegend; 1/500). Images were taken with a Leica DM5000B or Zeiss Axiovert 200M fluorescence microscope and processed using ImageJ 1.47n software (imagej.nih.gov/ij).
5
Flow Cytometry Analysis of T Cell Activation
6
Lamin-B1 Immunofluorescence with TSA
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The Biotin-XX-Tyramide Superboost kit (Invitrogen, USA, #2005939) was used for the TSA reaction. The rabbit anti lamin-B1 antibody (Abcam, USA, #ab16048) and a Streptavidin conjugate to Alexa 488 (1:200, Biolegend, #405235) were used for fluorescence staining.
7
Immunohistochemical Analysis of Testicular Stem Cells
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The testicular tissue fragments of the experimental groups, in addition to the tracing of DiI, were subjected to immunohistochemistry after tissue processing. To confirm the nature of SSCs, spermatocyte and spermatozoa the PLZF protein [17 (link)], SCP3 protein [20 (link)] and the ACRBP protein [21 (link)] were detected, respectively. The procedure of immunocytochemistry was performed according to previous study [22 ]. Briefly, tissues fixed with 4% paraformaldehyde (Sigma, USA) in PBS were Cryo-embedded in OCT compound (optimal cutting temperature) (Sakura, Japan) and cut into 5 µm-thick sections. Incubation with primary antibodies was applied for overnight at 37°. Then the second antibody was applied for 2 h at room temperature in the dark. Nuclei were counterstained with DAPI. Specimens were observed with a confocal laser microscope (TE 2000, Nikon, Japan). The following antibodies were used as primary antibodies: mouse anti PLZF antibody (1:100 Santa Cruz Inc, USA), Rabbit anti SCP3 antibody (1:400 Abcam, UK), Rabbit anti ACRBP antibody (1:300 Abcam, UK). The secondary antibodies used were goat anti mouse IgG and goat ant rabbit igG, conjugated with Alexa 488 (1:200, Bio legend UK).
8
Flow Cytometry Analysis of T Cell Activation
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2 × 106 cells were washed in FACS buffer (PBS, 10% FBS, and 0.05% sodium azide), and then resuspended in FACS buffer to a concentration of 1 × 106 cells/mL to probe GFP/YFP expression. For surface staining, primary CD4+ T cells were washed in FACs buffer, and then stained with anti-CD44 or anti-CD69 conjugated to Alexa-fluor 647 (Biolegend), CD45RO conjugated to PE-Cy5 (BD Pharmingen). For CD4 staining, cells were first stained with primary anti-CD4 (clone RPA-T4, Biolegend) and then with secondary Alexa 488 (Biolegend). Cells were left on ice for 30 min during staining while gently vortexing every 10 minutes. Cells were washed, and the mean fluorescence intensity (MFI) of each sample was obtained using Accuri C6 flow cytometer.
9
Lamin-B1 Diffusion Dynamics Quantification
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Cells from the completed cTSA-seq reaction were resuspended in PBS containing 0.001% Tween-20 and Streptavidin conjugated to Alexa 488 (1:200, Biolegend, #405235) and an anti-rabbit secondary conjugated (1:1000) to an Alexa 594 fluorophore. The cells were then stained with DAPI and mounted in Prolong anti-fade gold for imaging on a Leica SP5 scanning confocal microscope using the Leica Application Suite v2.7.3.9723. We used a 40× 1.4NA objective with Leica Type F immersion oil. Diffusion of the Streptavidin signal from the lamin-B1 source signal was measured using the lineplot feature in FIJI v2.1.0/1.53c. The iterative Levenberg-Marquardt algorithm (R package minpack.lm v1.2-1) was used to fit the fluorescence signal to the equation: y = y0 + A * exp(R0 * x).
10
Detecting Glycophorin A and PfLDH on EVs
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Isolated EVs were labeled with fluorescence antibodies to determine the presence of Glycophorin A coupled with Alexa 488 (Biolegend, California, United States) and PfLDH (LS-C488831, Life Span Biosciencies, China), which was coupled with APC/Cy7 following the instructions of the conjugation kit (ab102859, Abcam, Cambridge, UK). We first filtered both primary and secondary antibodies with 0.2 µm filters to reduce possible false detection due to antibody aggregation. EVL and EVH were permeabilized with 0.05% saponin in PBS for 10 min. Then samples from cultures were incubated 3 h with primary antibodies at a dilution of 1:200 and then ultracentrifuged at 110,000 × g for 2 h. This last step was repeated to wash the EV pellet after which the supernatant was discarded. The pellet was resuspended in 100 µl of double-filtered PBS 1X and analyzed by flow cytometry.
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