Purified recombinant proteins were labeled using amine reactive NHS ester dyes (Thermo Fisher Scientific). The Alexa fluorophores used to label proteins were Cdc15 – Alexa 488 (A-20000, Thermo Fisher); Fic1 – Alexa 647 (A-20006, Thermo Fisher); Pxl1 peptide (residues 177–189) – Alexa 405 (A30000, Thermo Fisher); and Cdc12 peptide (residues 20–40) – Alexa 405 (A30000, Thermo Fisher). All proteins and peptide were in 50 mM Tris, pH 7.4, 500 mM NaCl, 1 mM TCEP before the addition of dye. After addition of the dyes (dyes: protein molar ratio was 1:1), solutions were incubated for 1 hr on ice and excess dye was removed using Pierce Zeba Desalt Spin Columns (Thermo Scientific; 89882). The labeling efficiency was determined by measuring the protein concentration using Pierce BCA Protein Assay Kit (Thermo Scientific; 23225) before and after labeling with dyes.
Alexa 405
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom, Switzerland
Alexa-405 is a fluorescent dye used in various laboratory techniques. It has an excitation maximum at 401 nm and an emission maximum at 421 nm. The dye can be used for labeling proteins, nucleic acids, and other biomolecules for detection and analysis purposes.
Automatically generated - may contain errors
Lab products found in correlation
Alexa 488, by Thermo Fisher Scientific (14 mentions)
Alexa 647, by Thermo Fisher Scientific (13 mentions)
Alexa 594, by Thermo Fisher Scientific (7 mentions)
Axio observer z1, by Zeiss (6 mentions)
Alexa 555, by Thermo Fisher Scientific (5 mentions)
Vectashield, by Vector Laboratories (4 mentions)
Lsm 700, by Zeiss (4 mentions)
Ab13970, by Abcam (3 mentions)
Alexa 546, by Thermo Fisher Scientific (3 mentions)
Cyanine3 (cy3), by Jackson ImmunoResearch (3 mentions)
Alexa 568, by Thermo Fisher Scientific (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
Alexa 633, by Thermo Fisher Scientific (2 mentions)
Sc 10368, by Santa Cruz Biotechnology (2 mentions)
A0564, by Agilent Technologies (2 mentions)
Ab28364, by Abcam (2 mentions)
Tcs spe, by Leica camera (2 mentions)
Fluorescein isothiocyanate (fitc), by Jackson ImmunoResearch (2 mentions)
G2654, by Merck Group (2 mentions)
Gw10064f, by Merck Group (2 mentions)
27 protocols using alexa 405
1
Fluorescent Protein Labeling Protocol
2
Immunofluorescent Labeling of Larval Brains
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Larval brains were dissected in PBS and fixed for 20 min at room temperature in 4% formaldehyde and fixation buffer (PBS, 5 mM MgCl2, 0.5 mM EGTA). After fixation, brains were rinsed and washed in 0.3% PBS Triton X100 (PBT). Samples were blocked with 10% normal goat serum (NGS) in 0.3% PBT at room temperature and incubated with the primary antibodies overnight at 4°C. Brains were then washed in 0.3% PBT and incubated with the secondary antibodies overnight at 4°C. Brains were washed in 0.3% PBT and mounted in Vectashield (Vector Laboratories, Burlingame, CA, USA). The following primary antibodies and dilutions were used: guinea pig anti-Dpn (1:10,000) and rat anti-L’sc (1:5,000) (Caygill and Brand, 2017 (link)), chicken anti-GFP (1:2,000) from Abcam, mouse anti-Delta (1:50, C594.9B) from DSHB and mouse anti-Notch (intracellular domain, ICD) (1:50, C17.9C6) from DSHB. Fluorescently conjugated secondary antibodies Alexa405, Alexa488, Alexa546 and Alexa633 (all 1:200) from Life Technologies.
Images were acquired with a Leica TCS SP8 confocal microscope (Leica Microsystems, Wetzlar, Germany) and analysed with Fiji (Schindelin et al., 2012 (link)). Figures and illustrations were assembled using Adobe Photoshop CS3 and Adobe Illustrator CS3 (Adobe Systems, San Jose, CA, USA).
Images were acquired with a Leica TCS SP8 confocal microscope (Leica Microsystems, Wetzlar, Germany) and analysed with Fiji (Schindelin et al., 2012 (link)). Figures and illustrations were assembled using Adobe Photoshop CS3 and Adobe Illustrator CS3 (Adobe Systems, San Jose, CA, USA).
3
Detailed Immunofluorescence Staining Protocol
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Samples processes for immunofluorescence were performed as described previously 6 (link). Frozen sections were cut at 10 μm-thick. Primary antibodies used were: guinea pig anti-porcine insulin (A0564, DAKO, 1:600), chicken anti-insulin (GW10064F, Sigma, 1:1000), mouse anti-glucagon (G2654, Sigma, 1:1000), rabbit anti-glucagon (A0565, DAKO, 1:600), rabbit anti-somatostatin (A0566, DAKO, 1:600), rabbit anti-pancreatic polypeptide (T-4088, PenLabs, 1:750), goat anti-ghrelin (sc-10368, SantaCruz, 1:200), rabbit anti-GFP (Life Technologies, 1:500), chicken anti-GFP (ab-13970, Abcam, 1:500), guinea pig anti-Pdx1 (gift from C. Wright, 1:1000), rabbit anti-MafA (A300–611A, Bethyl, 1:500), rabbit anti-Nkx6.1 (BCBC, 1:400), rabbit anti-pHH3 (06–570, Upstate, 1:500), rabbit anti-CD31 (ab28364, abcam, 1:50), rabbit anti-Vimentin (ab92547, abcam, 1:100), rabbit anti-Tyrosine hydroxylase (ab152, chemicon, 1:1000), guinea pig anti-ARX(AB2834, BCBC, 1:100), rabbit anti-Synaptophysin (A0010, DAKO, 1:50). Secondary antibodies were coupled to Alexa 405, 488, 568, 647 (Life Technologies), FITC, Cy3, Cy5 (Jackson Immunoresearch), or TRITC (Southern Biotech). Sections were counterstained with DAPI. All sections were examined with a confocal microscope (Leica TCS SPE). In Fig. 2i , confocal tile-scan images were merged as a maximum projection.
4
Immunostaining of Chondrocyte Transcription Factors
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Primary cell cultures were fixed for 15 min at room temperature with 3% formaldehyde/PBS. The fixed cultures were washed and permeabilized with 0.1% Triton X‐100/PBS at room temperature for 10 min, incubated with primary antibodies diluted in PBS at room temperature for 1 hr, washed with PBS, and then incubated with regular secondary antibodies conjugated to Alexa 405 (Life Technologies) for 1 hr at room temperature. The following primary and secondary antibodies were used: Anti‐SOX9 antibody (Merck Millipore, AB5535), Anti‐SOX5 antibody (Abcam, ab94396), Anti‐SOX6 antibody (Abcam, ab30455), Anti‐p21 antibody (Cell Signaling Technology, 2946), Goat anti‐Rabbit IgG H + L Alexa Fluor 405 (Thermo Fisher, A‐31556), Goat anti‐mouse IgG H + L Alexa Fluor 405 (Abcam, ab175660).
5
Immunostaining of Glial and Neuronal Markers
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Antibodies against the following were used: Olig2 (#ab9610; RRID:AB_570666 ), SOX10 (#AB5727; RRID: AB_2195375 ), MBP (#SMI99; RRID: AB_2314772 ), NeuN (#MAB377; RRID: AB_2298772 ), PH3 (#05-806; RRID: AB_310016 ), and GAPDH (#MAB374, RRID: AB_2107445 ) from Millipore (Billerica, MA); GFAP (#Z0334; RRID: AB_10013382 ) from Dako (Copenhagen, Denmark); Iba1 (#019-19741; RRID: AB_839504 ) from Wako (Tokyo, Japan); caspase-3 (#9661; RRID: AB_2341188 ) from Cell signaling (Boston, MA); and A2B5 (#MAB312; RRID: AB_94709 ), O4 (#MAB345; RRID: AB_11213138 ), and O1 (#MAB344; RRID: AB_94860 ) from Sigma-Aldrich (St. Louis, MO). Alexa-405, Alexa-488, Alexa-555, and Alexa-637 secondary antibodies were obtained from Life Technologies. Dulbecco’s modified Eagle’s medium, 4′,6-diamidino-2-phenylindole, Neurobasal, and B27 supplements were obtained from Invitrogen (Carlsbad, CA). Triiodothyronine (#T6397) and other chemicals were obtained from Sigma-Aldrich unless stated otherwise.
6
Immunofluorescence Microscopy of PDAC Cells
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PDAC cells were fixed with 4% PFA for 15 min, then permeabilized with 0.2% Triton X-100 in PBS for 5 min, blocked for 5 min in 4% BSA in PBS. Primary antibodies were incubated 1 h in 4% BSA in PBS and detected with species-specific Alexa405, Alexa488, Alexa568 and Alexa647-conjugated secondary antibodies (Life Technologies). DAPI (Sigma) was used to stain nuclei. Coverslips were then mounted with Prolong Diamond Antifade Mountant (Invitrogen). Cells were imaged with a Zeiss 880 Laser Scanning Microscope with Airyscan with a Plan-Apochromat 63×/1.4 oil DIC M27 objective.
For colocalization analysis, the method was Adapted from Tyrrell et al., 2016 . The ImageJ function “Subtract Background” was used on the LPAR1 or Rab5/Rab11a/Rab7 channels. Then, a 15×15 μm box was placed over the center of the cell. The selected ROI was then analyzed using the Coloc2 plugin from ImageJ.
For colocalization analysis, the method was Adapted from Tyrrell et al., 2016 . The ImageJ function “Subtract Background” was used on the LPAR1 or Rab5/Rab11a/Rab7 channels. Then, a 15×15 μm box was placed over the center of the cell. The selected ROI was then analyzed using the Coloc2 plugin from ImageJ.
7
Immunohistochemistry Protocol for Drosophila Brains
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Brains were dissected in PBS, fixed in 4% formaldehyde/PBS for 20 min at room temperature and washed with PBS with 0.3% TritonX-100 (PBTx). Samples were blocked with 10% normal goat serum before overnight incubation with the following antisera: rabbit anti-Ase 1:2,000 (Brand et al., 1993 (link)) (a gift from the Jan lab), chicken anti-β-Galactosidase 1:1,000 (abcam ab9361), rabbit anti-β-Galactosidase 1:10,000 (Cappel), guinea pig anti-Dpn 1:5,000 (Caygill and Brand, 2017 (link)), rat anti-Elav 1:100 (DSHB, 7E8A10 conc.), chicken anti-GFP 1:2,000 (abcam ab13970), rabbit anti-PntP1 (1:500) (a gift from the Jim Skeath), mouse anti-Pros 1:30 (DSHB, MR1A), rabbit anti-Tll 1:300 (Kosman et al., 1998 (link)). Secondary antibodies conjugated to Alexa-405, Alexa-488, Alexa-546, Alexa-568, Alexa-633 all 1:500 (Life Technologies) or DyLight-405 1:200 (Jackson Laboratories) were used. Samples were mounted in Vectashield (Vector Laboratories) for imaging.
8
Sericin Labeling of Pancreatic Islets
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FITC-labeled sericin was diluted to a concentration of 0.1 % and added to the isolation solution used for pancreatic islet isolation. The obtained pancreatic islets were fixed in 4 % paraformaldehyde solution (paraformaldehyde; Wako) and excited at 480 nm using a confocal laser scanning microscope (TCS-SP2-AOBS; Leica).
Approximately 200 pancreatic islets that had been isolated in the same manner were collected to prepare 2-μm-thick sections (Murakami et al. 2000 (link)). One series of sections was stained for insulin (1:500; DAKO EnVision™ method) for light microscopic observation. Another series of sections was incubated with an Alexa 405 (1:100, goat polyclonal, anti-mouse IgG(H + L) secondary antibody; Life Technologies Corporation)-labeled anti E-cadherin antibody (1:100, mouse monoclonal, anti-E Cadherin IgG; abcam®, Cambridge, UK) and subjected to double immunofluorescence staining for FITC-labeled sericin and Alexa 405-labeled anti-E-cadherin. Alexa 405 and FITC were excited at 406 and 480 nm, respectively, and the sections were observed using a confocal laser scanning microscope.
Approximately 200 pancreatic islets that had been isolated in the same manner were collected to prepare 2-μm-thick sections (Murakami et al. 2000 (link)). One series of sections was stained for insulin (1:500; DAKO EnVision™ method) for light microscopic observation. Another series of sections was incubated with an Alexa 405 (1:100, goat polyclonal, anti-mouse IgG(H + L) secondary antibody; Life Technologies Corporation)-labeled anti E-cadherin antibody (1:100, mouse monoclonal, anti-E Cadherin IgG; abcam®, Cambridge, UK) and subjected to double immunofluorescence staining for FITC-labeled sericin and Alexa 405-labeled anti-E-cadherin. Alexa 405 and FITC were excited at 406 and 480 nm, respectively, and the sections were observed using a confocal laser scanning microscope.
9
Immunofluorescence of hIPSCs for Lineage Markers
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Immunofluorescence was conducted as described in Bulger et al. (2024) (link). hiPSCs were rinsed with PBS once, fixed in 4% paraformaldehyde (VWR) for 15-20 min, and subsequently washed three times with PBS. The fixed cells were permeabilized and blocked in a buffer comprised of 0.3% Triton X-100 (Sigma-Aldrich) and 5% normal donkey serum in PBS for one hour and then incubated with primary antibodies SOX2 (3579s; 1:200; Rb) CDX2 (ab157524; 1:500; Ms), or TBXT (AF2085; 1:400; Gt) diluted in antibody dilution buffer (0.3% Triton, 1% BSA in PBS) overnight. The following day, samples were washed three times with PBS and incubated with secondary antibodies in antibody dilution buffer at room temperature for 2 h. Secondary antibodies used were conjugated with Alexa 405, Alexa 555, or Alexa 647 (Life Technologies) at a dilution of 1:400. Cells were imaged at 10x, 20x, or 40x magnification on an inverted AxioObserver Z1 (Zeiss) with an ORCA-Flash4.0 digital CMOS camera (Hamamatsu).
10
Immunofluorescence Imaging of Pluripotency Markers
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Immunofluorescence was conducted as described in Bulger et al. 2023 (link). hiPSCs were rinsed with PBS 1X, fixed in 4% paraformaldehyde (VWR) for 15–20 minutes, and subsequently washed 3X with PBS. The fixed cells were permeabilized and blocked in a buffer comprised of 0.3% Triton X-100 (Sigma Aldrich) and 5% normal donkey serum in PBS for one hour and then incubated with primary antibodies SOX2 (3579s; 1:200; Rb) CDX2 (ab157524; 1:500; Ms), or TBXT (AF2085; 1:400; Gt) diluted in antibody dilution buffer (0.3% Triton, 1% BSA in PBS) overnight. The following day, samples were washed 3X with PBS and incubated with secondary antibodies in antibody dilution buffer at room temperature for 2 hours. Secondary antibodies used were conjugated with Alexa 405, Alexa 555, or Alexa 647 (Life Technologies) at a dilution of 1:400. Cells were imaged at 10x, 20x, or 40x magnification on an inverted AxioObserver Z1 (Zeiss) with an ORCA-Flash4.0 digital CMOS camera (Hamamatsu).
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