To visualize the binding of sEC1–2 to cells expressing hantavirus Gn/Gc, we infected U2OS cells with rVSVs bearing ANDV or HTNV Gn/Gc. At 12–14 h post-infection, cells were washed with cold PBS, and blocked with cold 10% FBS in PBS at 4 °C for 30 min. Cells were then stained for surface expression of viral Gn/Gc with mouse mAb anti-hantavirus Gn/Gc 1E11/D3 followed by anti-mouse Alexa Fluor 568 (Thermo Fisher) for 1 h each at 4 °C. In parallel, rVSV-ANDV/HTNV Gn/Gc-infected cells were incubated with purified Flagtagged sEC1–2 (5 μM for 1 h at 4 °C, and then washed extensively with PBS. To visualize bound sEC1–2, we fixed cells with 4% formaldehyde (Sigma-Aldrich), and stained them with anti-Flag M2 mouse mAb (Sigma-Aldrich) and secondary antibody anti-mouse Alexa Fluor 568 (Thermo Fisher) for 1 h each at 4 °C. To test the ability of mAb 3305 to block sEC1–2 binding to hantavirus Gn/Gc expressing cells, we preincubated 200 nM sEC1–2 with either control human IgG or mAb 3305 (5–500 nM) for 1 h at 4 °C and performed immunostaining for sEC1–2 as described above. Cells were visualized by fluorescence microscopy.
Anti mouse alexa fluor 568
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Anti-mouse Alexa Fluor 568 is a fluorescent secondary antibody that recognizes mouse primary antibodies. It is designed for use in various immunostaining and detection techniques, such as Western blotting, immunohistochemistry, and flow cytometry. The Alexa Fluor 568 dye provides a bright red fluorescent signal that can be detected using standard rhodamine filter sets.
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Lab products found in correlation
Alexa fluor 488 anti rabbit, by Thermo Fisher Scientific (19 mentions)
Anti mouse alexa fluor 488, by Thermo Fisher Scientific (8 mentions)
Alexa fluor 568 anti rabbit, by Thermo Fisher Scientific (7 mentions)
Hoechst 33342, by Thermo Fisher Scientific (5 mentions)
Triton x 100, by Merck Group (4 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (3 mentions)
Anti mouse alexa fluor 647, by Thermo Fisher Scientific (3 mentions)
Anti rabbit alexa fluor 647, by Thermo Fisher Scientific (3 mentions)
Mas coated slides, by Matsunami (2 mentions)
Zen software, by Zeiss (2 mentions)
Goat anti mouse hrp, by Thermo Fisher Scientific (2 mentions)
A5228 clone1a1, by Merck Group (2 mentions)
Dako mounting medium, by Agilent Technologies (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Roche (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Lsm 880, by Zeiss (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Arginase 1, by Santa Cruz Biotechnology (2 mentions)
Cd11b apc cy7, by BD (2 mentions)
Ccr2 fitc, by BioLegend (2 mentions)
44 protocols using anti mouse alexa fluor 568
1
Visualizing Hantavirus Glycoprotein Binding
2
Immunofluorescence Staining of Mouse Kidneys
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Mouse kidneys were fixed with the 4% paraformaldehyde solution (pH 7.2) overnight at 4°C, washed with phosphate buffered saline (PBS), cryoprotected with 10% and 20% sucrose at 4°C, embedded in OCT compound (Miles), and frozen in hexane at −80°C. Frozen sections (5 μm thick) were collected onto MAS‐coated slides (Matsunami Glass IND, Ltd.) and air‐dried (Ohkido, Segawa, Yanagida, Nakamura, & Miyamoto, 2003 ; Segawa et al., 2005 ). Serial sections were incubated with primary antibodies overnight at 4°C. Alexa Fluor 488 anti‐rabbit (Molecular Probes) and Alexa Fluor 568 anti‐mouse (Molecular Probes) were used as secondary antibodies for 60 min at room temperature. Thereafter, the sections were mounted with Dapi Fluoromount GTM (Southern Biotech).
3
Immunofluorescence Microscopy of Mouse Kidneys
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Mouse kidneys were fixed with the 4% paraformaldehyde solution (pH 7.2), overnight at 4 °C, washed with PBS, cryoprotected with 10% and 20% sucrose at 4 °C, embedded in Tissue-Tek O.C.T. Compound (Sakura Finetek Japan Co. Ltd., Tokyo, Japan), and frozen in hexane at − 80 °C. Frozen sections (5 μm thick) were collected onto MAS-coated slides (Matsunami Glass IND, Ltd., Osaka, Japan) and air-dried44 (link). For immunofluorescence microscopy, serial frozen sections were incubated with primary antibodies overnight at 4 °C. Alexa Fluor 488 anti-rabbit (Molecular Probes, Eugene, OR, USA) and Alexa Fluor 568 anti-mouse (Molecular Probes) were used as secondary antibodies for 60 min at room temperature44 (link),45 (link),47 (link)–49 (link). Thereafter, the sections were mounted with DAPI Fluoromount-G (SouthernBiotech, Birmingham, UK). Images were taken with an A1R confocal laser scanning microscope system (Nikon, Tokyo, Japan).
4
Immunostaining of C. elegans Embryos
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Immunostaining was performed using a methanol–4% formaldehyde fix as described previously (Skop and White, 1998 (link)) using the primary antibodies anti–ATX-2 mouse monoclonal (1:100; Ciosk et al., 2004 (link)), anti-GFP rabbit polyclonal (1:100; ab6556; Abcam, Cambridge, United Kingdom), anti–ZEN-4 rabbit serum (1:150; gift from Michael Glotzer, University of Chicago), and anti-actin [C4] mouse monoclonal (1:200; 691002; MP Biomedicals, Santa Ana, CA) diluted in PBSB (1× phosphate-buffered saline [PBS], 1% bovine serum albumin) and incubated overnight at 4°C. Unbound primary antibodies were removed by three washes for 5 min each with 1× PBS plus 0.5% Tween-20. Secondary antibodies used over the course of several different experiments were applied as follows: Alexa Fluor 488 anti-mouse (A-11001; Molecular Probes, Eugene, OR), Alexa Fluor 568 anti-mouse (A-11004; Molecular Probes), and Alexa Fluor 488 anti-rabbit (A-11008; Molecular Probes), with all secondary antibodies diluted 1:200 in PBSB. After 2 h of incubation in the dark at ambient conditions, unbound secondary antibodies were removed as described. The fixed and stained embryos were mounted with 8 μl of Vectashield with 4′,6-diamidino-2-phenylindole (DAPI; H-1200; Vector Laboratories, Burlingame, CA). Confocal imaging was performed on a departmental Zeiss 510 Confocal LSM operated with ZEN software (Carl Zeiss).
5
Immunofluorescence Staining of Neurons
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Neurons were rinsed in prewarmed HBSS and then fixed in warm 4% paraformaldehyde (PFA), 0.15% glutaraldehyde, and 0.1% Triton X-100, in BRB80 (80 mM PIPES, 1 mM EGTA, 1 mM MgCl2, pH 6.9) at 37°C for 10 minutes and then room temperature (RT) for 10 more minutes. Cells were rinsed twice in PBS, incubated with 5–10 mg/mL sodium borohydride in PBS for 15 minutes, rinsed, and then permeabilized with 0.4% Triton X-100 in PBS for 5 minutes. Cells were rinsed with PBS 3 times and incubated in blocking buffer (5% normal goat serum, 0.2% sodium azide, 0.1% Triton X-100 in PBS) for 1 hour at RT or overnight at 4°C. Primary antibodies diluted in blocking buffer were applied for 2 hours at RT. After three 5-minute washes in PBS + 0.05% Tween, secondary antibodies were applied for 1 hour at RT; then coverslips were washed and mounted. Primary antibodies used: rabbit anti-TUBB3 (BioLegend; catalog 802001), anti-mouse tyrosinated tubulin (MilliporeSigma; T5168), anti-mouse acetylated tubulin (Abcam; ab24610). Secondary antibodies used: Cy5–anti-rabbit (Jackson ImmunoResearch; 111-175-144), Alexa Fluor 568–anti-mouse (Invitrogen, Thermo Fisher Scientific; A11004). Actin was stained with phalloidin–Alexa Fluor 488 (Invitrogen, Thermo Fisher Scientific; A12379).
6
Immunological Profiling of Cancer Cells
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All antibodies with working dilution ratio, company source and catalog number are listed as follows: anti-E-cadherin (1:1000 for western blotting (WB) and 1:200 for immune fluorescent staining (IF); BD Biosciences; BD610182), anti-CDKN2A (1:1000 for WB; Abcam; ab3642), anti-EGFP (1:1000 for WB; Invitrogen; MA1-952) anti-β-actin (1:5000 for WB; Sigma; A5441); anti-p16 (1:100 for IF; BOSTER; BM1592). Secondary antibodies are listed as follows: Alexa Fluor 568 anti-mouse (1:500; Invitrogen; A11031), Alexa Fluor 488 anti-mouse (1:500; Invitrogen; A11029) and Alexa Fluor 488 anti-rabbit (1:500; Invitrogen; A11034). Anti-rabbit IgG HRP (1:3000; CST; #7074), anti-mouse IgG HRP (1:3000; CST; #7076). Alexa Fluor 568 Phalloidin (1:200 for F-actin labeling; Invitrogen; A22287). CellTracker Green (1:1000 for OPSCC cells or neutrophils labeling; Invitrogen; C7025), CellTracker Red (1:1000 for neutrophils labeling; Invitrogen; C34552). DAPI (4,6-Diamidino-2-phenylindole, 1:1000 for nuclei labeling; Sigma; D8417).
7
Comprehensive Antibody Validation Protocol
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The primary antibodies with working dilution factors, company source and catalog number include: anti-PCDH7 (1:500 for western blot (WB) and 1:200 for immunofluorescence (IF); Abcam; ab139274), anti-E-cadherin (1:1000 for WB; BD; 610181), anti-E-cadherin (1:200 for IF; CST; #3195S),anti-α-catenin (1:500 for WB; BD; 610193), anti-β-catenin (1:2000 for WB; BD; 610154), anti-γ-catenin (1:4000 for WB; BD; 610253), anti-p120-catenin (1:1000 for WB; BD; 610133), anti-pMLC2 (1:1000 for WB and 1:200 for IF; CST; #3671S), anti-PP1α(1:1000 for WB, 1:200 for IF and 1:100 for IP; Santa Cruz; sc-7482), anti-MLC2 (1:1000 for WB; CST; #3672s), anti-α-tubulin (1:1000 for WB; Proteintech; 11224-1-AP), anti-Flag-Tag (1:125 for Co-immunoprecipitation; Abbkine; BB-A02010), anti-Flag-Tag (1:500 for WB; Proteintech; 20543-1-AP), anti-mCherry (1:4000 for WB; GeneTex; GTX128508), anti-IgG (1:100 for IP; Abcam; ab102455). Secondary antibodies include Alexa Fluor 568 anti-mouse (1:500; Invitrogen; A11031), Alexa Fluor 568 anti-rabbit (1:500; Invitrogen; A11036), Alexa Fluor 647 anti-mouse (1:500; Invitrogen; A21236) and Alexa Fluor 647 anti-rabbit (1:500; Invitrogen; A21245). anti-rabbit IgG HRP (1:3000; CST; #7074), anti-mouse IgG HRP (1:3000; CST; #7076). Hoechst 33342 (H3570, Thermo) was used for cell nuclear staining. Y27632 (ROCK inhibitor, TOCRIS) was used at working concentration of 10 μM.
8
Validating α-Synuclein Pathology in Organotypic Hippocampal Slices
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Organotypic hippocampal culture slices (OHCS) were created from C57BL/6J pups on post-natal day 7 according to Stoppini et al. [51 (link)]. In order to validate the ability of the mouse full-length (1-140) α-Syn PFF for seeding aggregation before using them for in vivo experiments, the PFF were injected into the dentate gyrus of the OHCS, as previously described [18 (link)]. Slices were fixed 7 days post injection (dpi), and stained for pathological aggregates using conformation-specific α-Syn antibody MJF-14 (rabbit mAb MJF-14-6-4-2, 1:25,000, Abcam #ab209538) and pSer129 (mouse mAb 11A5, 1:10,000, kindly provided by ImagoPharmaceuticals), as described previously [18 (link)]. Alexa Fluor 488 anti-rabbit and Alexa Fluor 568 anti-mouse (Invitrogen, #A11008 and #A11004, 1:2000) were used for detection, along with 4′,6-diamidino-2-phenylindole (DAPI, TH.GEYER, 5 µg/mL) for staining nuclei. As negative controls, C57BL/6J OHCS were injected with either sterile PBS or monomeric α-Syn and processed as above. Furthermore, α-Syn knockout OHCS were injected with human S129A PFF and processed as above.
9
Mitochondrial and Autophagic Dynamics Imaging
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Cells were seeded on coverslips pretreated with poly-Llysine (Sigma-Aldrich, P4707), fixed with paraformaldehyde (PFA; 4% w:v; Fluka-Chemical, 76240), permeabilized with Triton X-100 solution (0.1% v:v in phosphate-buffered saline 1X; Sigma-Aldrich, T9284), and stained with an anti-Tom20 antibody (BD Biosciences, 556432) to measure mitochondrial morphology or an anti-LC3B antibody (Sigma-Aldrich, L7543) to visualize autophagic structures. The cells were then incubated with Alexa Fluor 568 antimouse (Invitrogen, A11031) or Alexa Fluor 488 anti-rabbit (Invitrogen, A11008) secondary antibodies. Chromatin condensation was labeled with Hoechst 33342 (Ho, 2 µM; Sigma-Aldrich, 101174857). For reactive oxygen species (ROS) quantification, cells were stained in vivo with dihydroethidium (He, 5 µM; Invitrogen, D1168) for 15 min at 37 °C, and they were then fixed with PFA. To measure lysosome size, cells were labeled in vivo with Lysotracker Red DND-99 (LTR, 2 µM; Invitrogen, L7528) for 15 min at 37 °C and then fixed with PFA. Fluorescence images were analyzed with an inverted fluorescence microscope (IX51, Olympus) equipped with a camera (DP70, Olympus).
10
Immunofluorescence Imaging of Nuclear Proteins
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U-2 OS or HeLa cells were seeded on to glass coverslips and after the indicated treatments were fixed in 4% paraformaldehyde and permeabilized with 0.2% Triton X-100. Primary antibody incubations were performed at 4°C overnight with the indicated antibodies: NKRF (GeneTex, GTX105380), XRN2 (Bethyl Laboratories, A301-103A), fibrillarin (Abcam, ab5821 and ab4566), anti-FLAG M2 (Sigma–Aldrich, F1804). Secondary fluorescent antibodies were incubated for 1 h at room temperature (Alexa Fluor 488 anti-Rabbit, Thermo Fisher Scientific, A11008; Alexa Fluor 568 anti-Rabbit, Thermo Fisher Scientific, A11011; Alexa Fluor 488 anti-Mouse, Thermo Fisher Scientific, A11001; Alexa Fluor 568 anti-Mouse, Thermo Fisher Scientific, A11004). Finally, the nuclei were labelled with Hoechst 33342 (Thermo Fisher Scientific). Images were acquired on Zeiss 710 confocal microscope and analysed with ImageJ.
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