A sperm sample from patient AY0283 and two normal sperm samples fixed with 4% PFA were applied to slides, permeabilized with 0.1% Triton X-100 (BS084, Biosharp) for 10 min, and then blocked with 10% donkey serum for 2 h at 37°C. To analyze the location and expression of DNAH6 in spermatozoa and the changes in flagellar-associated and acrosome-associated proteins, anti-DNAH6 (rabbit, 1:100, ab122333, Abcam, Cambridge, UK), anti-DNAH1 (rabbit, 1:100, ab122367, Abcam), anti-DNAI2 (rabbit, 1:200, 17533-1-AP, Proteintech), anti-SPAG6 (rabbit, 1:200, HPA038440, Sigma-Aldrich), anti-RSPH1 (rabbit, 1:100, HPA017382, Sigma-Aldrich), and anti-AKAP4 (rabbit, 1:200, HPA020046, Sigma-Aldrich) antibody were co-incubated with monoclonal anti-acetylated-tubulin antibody (mouse, 1:500, T6793, Sigma-Aldrich) at 4°C for 16 h. Anti-ACTL7A (rabbit, 1:100, HPA021624, Sigma-Aldrich) and anti-acrosin antibodies (rabbit, 1:200, NBP2-14260, Novus) were also incubated separately at 4°C for 16 h. We used Alexa Fluor 488 anti-mouse antibody (1:800, Jackson, Lancaster, PA, USA), Alexa Fluor 594 anti-rabbit antibody (1:800, Jackson), and Hoechst 33 342 (1:500, Thermo Scientific) as secondary antibodies. The stained samples were observed with a laser scanning confocal microscope (LSM800, Carl Zeiss AG) in selected channels (Alexa Fluor 594, Alexa Fluor 488, and Hoechst 33 342).
Alexa fluor 488
Manufactured by Zeiss
Sourced in Germany
Alexa Fluor 488 is a fluorescent dye used in various laboratory applications. It has an excitation maximum at 488 nm and an emission maximum at 519 nm, making it suitable for detection and imaging with common fluorescence microscopy and flow cytometry equipment.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (4 mentions)
Alexa fluor 555, by Zeiss (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Lsm 510, by Zeiss (2 mentions)
Axio imager, by Zeiss (2 mentions)
Lsm 700 confocal microscope, by Zeiss (2 mentions)
Axiovert 200m, by Zeiss (2 mentions)
Ab122333, by Abcam (1 mentions)
Ab122367, by Abcam (1 mentions)
T6793, by Merck Group (1 mentions)
Hpa021624, by Merck Group (1 mentions)
Nbp2 14260, by Novus Biologicals (1 mentions)
Bs084, by Biosharp (1 mentions)
Alexa fluor 594, by Zeiss (1 mentions)
Hpa038440, by Merck Group (1 mentions)
Hpa017382, by Merck Group (1 mentions)
Hpa020046, by Merck Group (1 mentions)
Hoechst 33342, by Zeiss (1 mentions)
Lsm 800, by Zeiss (1 mentions)
Axio observer inverted microscope, by Zeiss (1 mentions)
7 protocols using alexa fluor 488
1
Sperm Flagellar and Acrosomal Protein Analysis
2
Quantifying Biv4.40 Binding to Sn in CHO Cells
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The binding capacity of myc-tagged Biv4.40 to Sn was assessed by immunofluorescent staining. Biv4.40 was added to Sn-expressing CHO cells and non-transfected CHO cells that served as negative controls (30 (link)). Cells were seeded onto coverslips and incubated at 37°C for 24 h. Biv4.40 was added to the cells on ice for 60 min at 1 μg/mL. Cells were fixed with 4% paraformaldehyde for 20 min at ambient temperature. After fixation, the secondary mouse-anti-myc antibody (R950-25, ThermoFisher Scientific) was added at 1 μg/mL. After three wash steps with PBS, the chicken anti-mouse Alexa Fluor 488 (ThermoFisher Scientific, A21200) antibody was added to the cells. Nuclei were stained with DAPI (Sigma-Aldrich). Images were obtained using an Axio Observer inverted microscope (Zeiss) equipped with a Compact Light Source HXP 120C and with filter sets 49 and 10 for DAPI and Alexa Fluor 488 fluorophores, respectively. Images were processed using Image J software (Supplementary Figures 1B,C ).
3
3D Spheroid Imaging and Analysis
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Images were acquired on the Zeiss Axio Observer Z1 microscope, using the 63 × Plan-Apochromat (NA 1.4) oil, the 40 × LD Plan-Neofluar (NA 0.6) and the 20 × Plan-Apochromat (NA 0.8) objectives, the AxioCam MRm Rev.3 camera, and the software package AxioVision version 4.8.2 (all from Zeiss, Göttingen, Germany). Filter sets and related staining were as follows: (1) Alexa Fluor 488—filter set 38 HE, (2) Alexa Fluor 555—filter set 43 HE, (3) Phalloidin-Alexa Fluor 660—filter set 50, and (4) DAPI—filter set 49 (all filter sets from Zeiss). Summary images of spheroids were acquired by fluorescence microscopy in four colors with 35 z-slices per spheroid. Classification of spheroids: Spheroid 3D structure, polarity and lumen formation was determined manually on blinded/masked images by three raters using 4-color z-stacks, as described previously [23 (link)], and discrepant rating discussed, before unmasking and calculation of distribution.
4
Immunofluorescence Staining of Cells
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Cells were seeded on collagen-coated (500 µg/mL) coverslips. The cells were then fixed with 5% formalin for 30 min, permeabilized with 0.1% Triton X-100 for 20 min, and preblocked with PBS containing 5% bovine serum albumin (BSA; Affymetrix, Santa Clara, CA, USA) and 1% normal goat serum (NGS; Vector Laboratories) for 1 h. The cells were then incubated with the indicated primary antibody overnight at 4 °C, followed by Alexa Fluor 488 (Life Technologies, Carlsbad, CA, USA) or Alexa Fluor 555 (Life Technologies) secondary antibodies for 4 h at 4 °C and counterstained with DAPI for 10 min at room temperature. After incubation, the cells were mounted in Gel/Mount media (Biomeda Corporation, Foster City, CA, USA). The fluorescence signal was visualized using a confocal microscope (LSM510; Carl Zeiss) at excitation wavelengths of 488 nm (Alexa Fluor® 488), 543 nm (Alexa Fluor® 555), and 405 nm (DAPI).
5
Fungal Staining in Root Tissue
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In order to stain fungal and oomycete structures within root tissue, Wheat Germ Agglutinin (WGA) conjugated to AlexaFluor488 (Life Technologies GmbH, Darmstadt, Hesse, Germany) was used enabling staining of cell wall components of fungi and oomycetes, such as A. euteiches. Freshly harvested roots were placed in 50% (v/v) ethanol for at least four hours followed by incubation in 20% KOH for 10 min at 90 °C. After washing with distilled water, roots were incubated in 0.1 M HCl for 2 h and then transferred into phosphate-buffered saline (135 mM NaCl, 3 mM KCl, 1.5 mM KH2PO4, 8 mM Na2HPO4, pH 7.4) containing 0.2 µg ml−1 WGA-AlexaFluor488 for at least 6 h. Stained roots were analyzed using an epifluorescence microscope AxioImager (Zeiss GmbH, Jena, Thuringia, Germany) using the reflector module 09 (EX BP 450-490/BS FT510/EM LP515). Photographs were taken by an AxioCam (Zeiss) and combined using Adobe Photoshop.
6
Visualizing Megalin Colocalization in Kidney
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To assess micelle colocalization with megalin, kidney tissue sections were first washed with Tris buffered saline (TBS) plus 0.025% Triton X‐100 with gentle agitation for 10 minutes. A block buffer with 1% bovine serum albumin (BSA), 10% normal goat serum, and 0.3 m glycine in 0.1% PBS Tween was applied to the slides for 1 hour at room temperature. Then, the slides were applied with an anti‐Lrp2/Megalin antibody (Abcam, Cambridge, UK, 1:100) overnight at 4°C. The following day, slides were rinsed twice with TBS plus 0.025% Triton for 5 minutes and applied with fluorophore‐conjugated secondary antibody‐goat, anti‐mouse IgG H&L Alexa Fluor® 488 (Abcam, Cambridge, UK, 1:1000) for 1 hour at room temperature in the dark. Slides were then counterstained with DAPI and mounted with VectaMount™ mounting medium (Vector Laboratories, Burlingame, CA). Fixed slides were imaged with a LSM 700 confocal microscope (Zeiss, Oberkochen, Germany), and the colocalization between Cy7 and Alexa Fluor® 488 channels was performed on ImageJ with coloc2.
7
Fluorescence Microscopy of Stressed Cells
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Standard procedure Cells were fixed and processed for fluorescence microscopy as described previously 81 . Briefly, cells were grown on glass coverslips, stressed as indicated, fixed using 3.7 % formaldehyde (Sigma) in PBS for 10 min, followed by 5 min post-fixation/permeabilization in ice-cold methanol. Cells were blocked for 1 h in 5% horse serum/PBS, and primary and secondary incubations performed in blocking buffer for 1 h. All secondary antibodies were raised in donkey against either mouse, rabbit or goat and tagged with either Alexa Fluor 488, 568 or 647 (Thermo Fisher). Following washes with PBS, cells were mounted in polyvinyl mounting media and viewed using a Zeiss Axiovert 200M with a 63X Plan Apochromat objective lens (NA 1.4) and illuminated with a mercury lamp and standard filters for DAPI (Filter Set 49: 335-383 / 395 / 420-470), Alexa Fluor 488 (Filter Set 10: 450-490 / 510 / 515-565), Alexa Fluor 568 (Filter Set 20: 540-552 / 560 / 575-640), Alexa Fluor 647 (Filter Set 50: 640/30 / 660 / 690/50). Images were captured using an AxioCam ICc5 digital camera (Zeiss) with the manufacturer's software, and raw TIF files were imported into Adobe Photoshop. Identical adjustments in brightness and contrast were applied to all images in each experiment.
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