All steps were carried out at room temperature, except when indicated otherwise. Chips were washed 3 times with PBS, fixed for 12 min with 4% PFA (Invitrogen, USA), and washed again 3 times with PBS. For cell permeabilization, samples were incubated in 0.1% Triton X-100 (Sigma-Aldrich, USA) for 10 min. After washing 3 times with PBS, blocking was done for 1 h with a 2% BSA in PBS solution. Then, the primary antibody (anti PECAM-1, Cell Signaling, USA) diluted 1:100 in blocking solution was added and incubated at 4 °C overnight. After washing 3 times with PBS, a secondary antibody (1:500, anti-mouse 488 or anti-mouse 546, Invitrogen, USA), Hoechst (1:1000, Sigma, USA), and phalloidin 670 (1:150, Acti-Stain, Cytoskeleton, USA) in blocking buffer were added and incubated for 2 h. The chips were washed 4 times with PBS and imaged on a confocal microscope (Zeiss LSM710 and LSM980, Zeiss, Germany).
Anti mouse 546
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Anti-mouse 546 is a fluorescent labeling reagent used in various biological and research applications. It is designed to specifically detect and visualize mouse-derived proteins or other biomolecules in samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti mouse 488, by Thermo Fisher Scientific (2 mentions)
Anti rabbit 568, by Thermo Fisher Scientific (2 mentions)
Anti rabbit 488, by Thermo Fisher Scientific (2 mentions)
Acti stain, by Cytoskeleton (1 mentions)
Hoechst, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (1 mentions)
Anti pecam 1, by Cell Signaling Technology (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Lsm 980, by Zeiss (1 mentions)
Anti mouse cy5, by Jackson ImmunoResearch (1 mentions)
Affigel, by Bio-Rad (1 mentions)
Mab302, by Merck Group (1 mentions)
Np 001098373.1, by Charles River Laboratories (1 mentions)
A21271, by Merck Group (1 mentions)
A10262, by Thermo Fisher Scientific (1 mentions)
Anti chicken 488, by Jackson ImmunoResearch (1 mentions)
Cd11b pe dazzle, by BioLegend (1 mentions)
Panck fitc, by Merck Group (1 mentions)
Prolong gold dapi, by Thermo Fisher Scientific (1 mentions)
A11001, by Thermo Fisher Scientific (1 mentions)
5 protocols using anti mouse 546
1
Immunofluorescence Staining of PECAM-1
2
Immunohistochemistry of Embryonic Tissue Sections
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For immunohistochemistry embryos were fixed at the indicated stages in 4% PFA in 1 x PTW over night at 4 °C. Fixed embryos were either heated (Inoue and Wittbrodt, 2011 (link)) and mounted for cryo-sectioning or stained whole mount according to the protocols adapted from the zebrafish book (Westerfield, 2000 ). In case of sectioning, frontal or lateral cryosections (as indicated) of 16 μm thickness were prepared and stained as described in (Inoue and Wittbrodt, 2011 (link)). The following primary antibodies were used: anti-GS (mouse, Chemicon MAB302, 1:500), anti-Rx2 (rabbit, self-made, 1:250, anti-olRx2 antibody was raised against the full-length olRx2 (NP_001098373.1) recombinant protein in rabbits (Charles River), and affinity purified using the antigen coupled to AffiGel (Biorad) (Herder et al., 2013 (link)), anti-GFP (chicken, life technologies A10262, 1:500) and for anti-HuCD (mouse, invitrogen A-21271, 1:250) and anti-acetylated Tubulin (mouse, SIGMA T7451-100UL, 1:100). The following secondary antibodies were used at a 1:500 dilution, anti-mouse Cy5 (Jackson 715-175-151); anti mouse 546 (invitrogen A-11030), anti chicken 488 (Jackson 703-545-155), anti rabbit 549 (Jackson 112-505-144). DAPI (SIGMA, D956) nuclear counterstaining was performed (Inoue and Wittbrodt, 2011 (link)).
3
Immunofluorescent Staining of Pancreas Tissue
4
Antibody-based Cellular Protein Detection
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Antibodies against P1 (Plasmatocytes, 1:50), L1 (Lamellocytes, 1:50), and C4 (Crystal cells, 1:50) were incubated for 1 h at room temperature in PBS and subsequently washed 3 × 10 min in PBS. For intracellular proteins, the samples were incubated overnight at 4°C in anti-pJNK (1:250), anti-Idgf3 (1:50), or anti-CC3 (1:400) diluted in PBST (1% TritonX-100) and subsequently washed 3 × 10 min in PBS. The samples were incubated with secondary antibody anti-mouse-546 (1:500, Thermo Fisher #A11030), anti-mouse-488 (1:250, Thermo Fisher #A11001), anti-rabbit-568 (1:500, Thermo Fisher #A21063), and anti-rabbit-488 (1:500, Thermo Fisher #A11008), or 4’,6-Diamidino-2-phenylindole dihydrochloride (DAPI) (1:500, Sigma-Aldrich D9542) for 1 h at room temperature and washed 3 × 10 min with PBS before mounting in Fluoromount-G (Thermo Fisher, 00-4958-02).
5
Immunofluorescence Analysis of ECM Proteins
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Antibodies against SPARC (1:3000:this report), Nidogen (1:2000) [53] , Laminin (1:2000) [54] and Perlecan (1:2000) [55] were incubated overnight (ON) at 4°C either in PBS (nonpermeabilized) or PBS-T (permeabilized). PPO1 (1:500), PPO2 (1:250), Tiggrin (1:500, [56] a kind gift by A. Simmonds, Alberta) were stained in PBS at 4°C O/N. Anti-ß-integrin (1:200, (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. DSHB #CF.6G11) and Anti-Dlg (1:50, DSHB #4F3, [44] ) was incubated at 4°C, O/N in PBS-T. Anti-HA (1:3000, Thermofisher #5B1D10) was pre-incubated with fixed Ras V12 salivary glands for 1h at RT in PBS-T and subsequently incubated with the samples for 1h at RT. Samples were washed 3 x 10 min with PBS/PBS-T and incubated with secondary antibody anti-Rabbit-568 (1:500, Thermofisher #A21069), anti-Rabbit-488 (1:100, Thermofisher #A11008) or anti-Mouse-546 (1:500, Thermofisher #A11030), DAPI (1:500, Sigma-Aldrich D9542) and Phalloidin-488/546 (1:500, Sigma-Aldrich #A12379 and # A22283, respectively) for 1 h at room temperature RT, washed 3 x 10 min with PBS or PBS-T before mounting in FluoromountG.
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