Chick metacarpals at E8 were dissected, fixed in 4% paraformaldehyde and frozen in O.C.T. (Tissue-Tek) for making sections (20 μm thickness). For col2a antibody staining, tissue sections were pretreated with 1 mg ml−1 pepsin in 0.1 N HCl and blocked in 10% fetal bovine serum before incubating with anti-col2a (1:50, DSHB) and further anti-mouse alexa-488 (1:250, Invitrogen). Phalloidin alexa-546 (1:100, Molecular Probes) was used for counterstaining. For section in situ hybridization, tissue sections were hybridized to anti-chick Ihh RNA probes labelled with digoxigenin. Tissue sections of the post-imaging chick metacarpal (expressing GFP) were stained with Phalloidin alexa-546 (1:100, Molecular Probes). The number of green (2483 PZ cells) and red signals (2560 PZ cells) of the illuminated half of the tissue was counted using ImageJ.
Alexa 546 phalloidin
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 546 phalloidin is a fluorescent dye used to label and visualize F-actin in fixed and permeabilized cells. It binds selectively to F-actin, enabling the detection and localization of the actin cytoskeleton.
Automatically generated - may contain errors
Lab products found in correlation
Mouse anti fasciclin 3, by Developmental Studies Hybridoma Bank (2 mentions)
Rabbit anti gfp, by Thermo Fisher Scientific (2 mentions)
Fluoromount g, by Southern Biotech (2 mentions)
A2163, by Merck Group (2 mentions)
Alexa 488, by Thermo Fisher Scientific (2 mentions)
Anti mouse alexa 488, by Thermo Fisher Scientific (1 mentions)
Normal igg, by Santa Cruz Biotechnology (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Fv1000 confocal microscopy, by Olympus (1 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (1 mentions)
Goat serum, by Merck Group (1 mentions)
Prolong glass, by Thermo Fisher Scientific (1 mentions)
Anti phospho c jun antibody, by Cell Signaling Technology (1 mentions)
Rat anti gfp, by Nacalai Tesque (1 mentions)
Cyanine3 (cy3), by Jackson ImmunoResearch (1 mentions)
Rat anti elav, by Developmental Studies Hybridoma Bank (1 mentions)
Mouse anti mmp1, by Developmental Studies Hybridoma Bank (1 mentions)
Mouse anti myc, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti gapdh, by Cell Signaling Technology (1 mentions)
Anti mouse igg irdye 800, by LI COR (1 mentions)
22 protocols using alexa 546 phalloidin
1
Chick Metacarpal Development Analysis
2
Immunofluorescence Staining of p120 and E-cadherin
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p120 siRNA (pool)and E-cadherin siRNA (pool) were purchased from Dharmacon. p120 cDNA was obtained from the University of Illinois at Chicago. All primary antibodies and normal IgG are purchased from Santa Cruz Biotechnology. 4′, 6-diamidino-2-phenyl indole dihydrochloride (DAPI), Alexa-labeled secondary antibodies and Alexa 546 phalloidin were purchased from Invitrogen.
3
Quantifying H. pylori-Induced c-Jun Activation
4
Immunofluorescence Staining Antibodies
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The following antibodies were used at the indicated dilutions: mouse anti-Fasciclin III 1:200 [7G10, Developmental Studies Hybridoma Bank (DSHB)], mouse anti-β-Galactosidase 1:200 (40-1A, DSHB), rabbit anti-GFP 1:1000 (Invitrogen, USA), rat anti-GFP (1:1000, Nacalai Tesque, Japan), Dylight-488 goat anti-rabbit IgG(H + L), Dylight-549 goat anti-mouse IgG(H + L) (Jackson ImmunoResearch Laboratories, USA), Alexa-546 Phalloidin 1:50 (Invitrogen). Yki rabbit polyclonal antibody was generated with full-length Yki29 (link). Rabbit anti-Cas (1:5,000) was generous gifts from Dr. Chang and Dr. Jang48 (link).
5
Immunohistochemistry and X-Gal Staining of Drosophila Larvae
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Tissues from third-instar larvae were processed as described previously (Külshammer and Uhlirova, 2013 (link)). The following primary and secondary antibodies were used at the indicated dilutions: mouse anti-MMP1 (mixture of 14A3D2, 3A6B4 and 3B8D12, 1:300), rat anti-ELAV (1:200; 7E8A10) and mouse anti-Fasciclin III (1:300, 7G10), all from Developmental Studies Hybridoma Bank (Iowa); and rabbit anti-Jun (1:500; present study). After washing, samples were incubated with a corresponding secondary antibody coupled to Cy3 or Cy5 (Jackson ImmunoResearch) for 2 h. Samples were counterstained with Alexa 546-phalloidin (Invitrogen) and DAPI to visualize actin filaments and nuclei, respectively. The lacZ activity was detected in imaginal discs using a standard X-Gal (5-bromo-4-chloro-3-indolyl-β-d -galactopyranoside) staining procedure described previously (Külshammer and Uhlirova, 2013 (link)).
6
Protein Expression Detection Methods
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Primary antibodies used were rabbit anti-Gas7 (Sigma), rabbit anti-Cux1 (Santa Cruze), rabbit anti-GFP (Invitrogen), mouse anti-actin-mCherry (Sigma), rabbit anti-GAPDH (Cell Signaling Technology) and mouse anti-MYC (Santa Cruze) and alexa-546 phalloidin (Invitrogen).
Secondary antibodies included anti-rabbit IgG IRDye 680 and anti-mouse IgG IRDye 800 (LICOR Bioscience) for western blot and Alexa 488 anti-rabbit IgG, alexa 555 anti-rabbit IgG (Invitrogen) for immunostaining.
Secondary antibodies included anti-rabbit IgG IRDye 680 and anti-mouse IgG IRDye 800 (LICOR Bioscience) for western blot and Alexa 488 anti-rabbit IgG, alexa 555 anti-rabbit IgG (Invitrogen) for immunostaining.
7
Phalloidin Staining of Embryos
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Embryos were fixed overnight in 4% paraformaldehyde in PBS at 4C. Following two washes for 5 min each in PBSTw (PBS + 0.1% Tween-20), embryos were manually dechorionated. To permeabilize, embryos were incubated for 2 hr in PBS + 2% Triton X100. Following two washes for 5 min each in PBSTw, embryos were blocked for 1 hr with 2% BSA in PBSTw. Embryos were incubated for 2 hr at RT in Alexa 546 phalloidin (Invitrogen) diluted 1:40 in 2%BSA/PBSTw. Following 6 washes for 10 min each in PBSTw, embryos were mounted in agarose for confocal microscopy.
8
Fluorescent Imaging of Cellular Actin and Apoptosis
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Alexa546-phalloidin, lipidTOX red, actin-GFP and carboxy-H2DFFDA were from Thermo Fisher Scientific. AAPH was from Cayman Chemical. Rabbit anti-ABCA1 was from Novus Biologicals. Antibodies to active Caspase-3 and GAPDH were from Abcam. Filipin, mevinolin, N-acetyl-l-cysteine (NAC), MβCD, water-soluble cholesterol (CHOL) and all other chemicals were from Sigma. KRBH buffer (128.8 mM NaCl, 4.8 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 2.5 mM CaCl2, 5 mM NaHCO3, 10 mM HEPE, pH 7.4) was used for all experiments.
9
Transfection of Recombinant M10 Constructs
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The recombinant M10 constructs were transfected into HEK293 or HeLa cells (no differences were observed in results for these two cell types). Cells were plated onto glass coverslips and transfected using either FuGENE 6 (Roche Diagnostics, Burgess Hill, West Sussex, UK) or Gene-juice (Millipore Ltd., Watford, Herts, UK) following the recommended protocols. 12–16 h later, the cells were either fixed and then stained for actin using Alexa-546 phalloidin (ThermoFisher Scientific, Hemel Hempstead, HERTS, UK) or prepared for live-cell imaging.
10
Structural Analysis of Eyelid and Tarsal Plate
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The eyelids or tarsal plates were incubated in permeabilization buffer (0.25% dimethyl sulfoxide [DMSO], 0.25% Triton X-100, and 15% sucrose in PBS) for 30 minutes at room temperature under constant rotation and rinsed with PBS. Thereafter, the eyelids were stained with Alexa-546 Phalloidin (Thermo Fisher Scientific, Wilmington, DE, USA) and 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich Corp.) in staining buffer (0.25% DMSO, 0.01% saponin, and 15% sucrose in PBS) overnight under constant rotation at 4°C protected from light. Tarsal plates were stained with biotinylated HA binding protein (HABP-385911; Millipore, Billerica, MA, USA) in staining buffer (0.25% DMSO, 0.01% saponin, and 15% sucrose in PBS) overnight under constant rotation at 4°C followed by incubation of the NeutrAvidin Alexa Fluor 555 conjugate overnight under constant rotation at 4°C protected from light. The eyelids and tarsal plates were washed with staining buffer followed by PBS and mounted on glass slides in Fluoromount-G (SouthernBiotech., Birmingham, AL, USA). The eyelids were imaged using an LSM 800 confocal microscope with Axiocam 503 color (Zeiss, Oberkochen, Germany). For analyzing the distribution of HA matrices, the tarsal plate whole mounts were imaged with Aryscan by using the 63× objective.
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