NETs were stained as previously described5 . Briefly, after blocking and permeabilization, neutrophils were incubated with a mouse monoclonal antibody (IgG2a) against DNA/Histone 1 (MAB3864; Millipore 0.55 mg/ml, diluted 1:1000) for 1 h at room temperature. As secondary antibodies a goat anti-mouse antibody (Dye Light488 conjugated highly cross-absorbed, Thermo Scientific; diluted 1:500 in blocking buffer) and goat-anti-rabbit antibody (Alexa633, Thermo Scientific; diluted 1:500 in blocking buffer) were used. To detect PR-39 a polyclonal rabbit anti‐PR‐39 antibody (1:50) was used as previously described5 . To detect A.pp a polyclonal rabbit anti‐A.pp St7 antibody (1:50) was used. As secondary antibody goat-anti-rabbit (Alexa633, Thermo Scientific; diluted 1:500 in blocking buffer) was used. Samples were recorded using a Leica TCS SP5 AOBS confocal inverted-base fluorescence microscope with a HCX PL APO ×40 0.75–1.25 oil immersion objective.
Alexa 633
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa 633 is a fluorescent dye used in a variety of biological applications. It emits light in the far-red region of the visible spectrum, making it suitable for use in techniques such as flow cytometry, fluorescence microscopy, and immunoassays.
Automatically generated - may contain errors
Lab products found in correlation
Alexa 488, by Thermo Fisher Scientific (33 mentions)
Alexa 568, by Thermo Fisher Scientific (11 mentions)
Alexa 555, by Thermo Fisher Scientific (10 mentions)
Alexa 546, by Thermo Fisher Scientific (7 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (6 mentions)
Goat anti rabbit igg, by Thermo Fisher Scientific (6 mentions)
Goat anti mouse igg, by Thermo Fisher Scientific (6 mentions)
Vectashield, by Vector Laboratories (5 mentions)
Rabbit anti gfp, by Thermo Fisher Scientific (5 mentions)
Tcs sp8 confocal microscope, by Leica camera (5 mentions)
Alexa 594, by Thermo Fisher Scientific (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Alexa 405, by Thermo Fisher Scientific (2 mentions)
Lsm 510 meta confocal microscope, by Zeiss (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (2 mentions)
Vectashield with dapi, by Vector Laboratories (2 mentions)
Dylight 405, by Jackson ImmunoResearch (2 mentions)
Propidium iodide, by Merck Group (2 mentions)
Normal goat serum (ngs), by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
78 protocols using alexa 633
1
Immunofluorescence Staining of NETs
2
Immunostaining of Imaginal Discs
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Imaginal discs were prepared and stained using standard procedures. Briefly, larvae were dissected in cold PBS and fixed in 4% paraformaldehyde (PFA) in PBS for 20 min. Washes were performed in PBS + 0.03% Triton X-100 (PBT) and blocking in PBT + 2% normal donkey serum (PBTN). Samples were incubated with primary antibodies overnight at 4˚C. The primary antibodies used: mouse anti-Wingless (1:2000, was deposited to the DSHB by Cohen, S.M.
(DSHB Hybridoma Product 4D4)), rat anti-DE-Cadherin (1:30, was deposited to the DSHB by Uemura, T. (DSHB Hybridoma Product DCAD2)), rabbit anti-phospho-Myosin light chain 2 (Ser19) (1:50, Cell Signaling Technology #3671), rabbit anti-Ap (1:1000, described in [52] ).
Incubation with secondary antibodies were at room temperature, for 2hr. The secondary antibodies used: anti-mouse Alexa 568 (1:700, ThermoFisher) and Alexa 633 (1:700, ThermoFisher), anti-rat Cy3 (1:300, Jackson ImmunoResearch) anti-rabbit Alexa 568 (1:600, ThermoFisher) and Alexa 633 (1:600, ThermoFisher). Discs were mounted in Vectashield antifade mounting medium with Dapi (Vector Laboratories). For F-actin staining Phalloidin-Tetramethylrhodamine B (Fluka #77418) was added during incubation with secondary antibodies at the concentration 0.3 μM.
(DSHB Hybridoma Product 4D4)), rat anti-DE-Cadherin (1:30, was deposited to the DSHB by Uemura, T. (DSHB Hybridoma Product DCAD2)), rabbit anti-phospho-Myosin light chain 2 (Ser19) (1:50, Cell Signaling Technology #3671), rabbit anti-Ap (1:1000, described in [52] ).
Incubation with secondary antibodies were at room temperature, for 2hr. The secondary antibodies used: anti-mouse Alexa 568 (1:700, ThermoFisher) and Alexa 633 (1:700, ThermoFisher), anti-rat Cy3 (1:300, Jackson ImmunoResearch) anti-rabbit Alexa 568 (1:600, ThermoFisher) and Alexa 633 (1:600, ThermoFisher). Discs were mounted in Vectashield antifade mounting medium with Dapi (Vector Laboratories). For F-actin staining Phalloidin-Tetramethylrhodamine B (Fluka #77418) was added during incubation with secondary antibodies at the concentration 0.3 μM.
3
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).
4
Nef-TagRFP657 Localization Assay
5
Immunostaining of Drosophila Wing Discs
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Wing discs were dissected, stained and fixed according to standard protocols. Primary antibodies used were rat anti-DE-cad (DCAD2, Developmental Studies Hybridoma Bank (DSHB) 1:50) and mouse anti-Wingless (4D4, DSHB, 1:100). Secondary antibodies were donkey anti-rat CY5 IgG (H+L) (Jackson ImmunoResearch Laboratories, 1:200) and anti-mouse Alexa 633 (Life Technologies, 1:200).
6
Immunofluorescence Staining of Cochlear Organelle
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Mice were anesthetized and killed, and inner-ear tissues were then removed. The cochleae were further dissected and fixed in 4% paraformaldehyde. Immunofluorescence staining with antibodies against CAV2 (mouse IgG; BD) and Prestin (goat IgG; Santa Cruz) along with DAPI (Vector Laboratories) was performed on whole-mount preparations of the carefully resected organ of Corti or cochlear cryosections (7 μm). We incubated the tissues in the antibody solutions for 1 h at room temperature after blocking with 2% bovine serum albumin in phosphate-buffered saline. Fluorescence confocal images were obtained with a LSM510-META confocal microscope (Carl Zeiss). CAV2 was labeled with Alexa 488 (mouse IgG; Life Technologies) and observed by confocal laser microscopy using a 488-nm laser. Prestin was labeled with Alexa 633 (mouse IgG; Life Technologies) and observed by confocal microscopy using a 633-nm laser. Some of the red signals in the figures reflect the pseudo-color of the Alexa 488 signal.
7
Imaging HO-1 Expression in SH-SY5Y Cells
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To study HO-1 expression, SH-SY5Y cells were grown as wild type or differentiated in 8-well chamber slides and then exposed to 500 μM H2O2 or 50 μM tBHQ for 24 h. By means of a standard technique of immunofluorescence (fixing in cold methanol), HO-1 expression was detected by using anti HO-1 (10 μg/ml rabbit anti HO-1, Origene) and ALEXA 633 (anti rabbit 1:400, Life-Technologies). Images were collected by using a three-channel TCS SP2 laser scanning confocal microscope (Leica Mycrosystems).
8
Immunostaining of the Notum in Drosophila
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Dissection and immunostainings of nota were performed as indicated in ref. 68 (link) with standard formaldehyde fixation and permeabilisation/washes in PBT 0.4% Triton. The following antibody was used: mouse anti-acetylated-Tubulin (1/200, Sigma T7451, clone 611B1). A secondary antibody was produced in goat with Alexa 633 (1/100, Life technologies, A21052, lot 1712097). Dissected nota were mounted in Vectashield with DAPI (Vectorlab).
9
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.
10
Immunohistochemistry of Zebrafish Connexins
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Anesthetized embryos from 2 to 14 dpf were fixed in either 2% trichloroacetic (TCA) acid for 3 hr or 4% paraformaldehyde (PFA) for 1 hr. Fixed tissue was then washed in PBS + 0.5% TritonX100, followed by standard blocking and antibody incubations. Tissue was cleared step-wise in a 25%, 50%, 75% glycerol series and was dissected and mounted for imaging. Primary antibodies used were: chicken anti-GFP (abcam, ab13970, 1:250), rabbit anti-human-Cx36 (Invitrogen, 36–4600, 1:200), rabbit anti-Cx34.7 against the intracellular loop peptide of the perch protein (JOB, 1:20)(O'Brien et al., 2004 (link)), mouse anti-Cx35.1 against recombinant perch protein (EMD/Millipore, MAB3045, 1:200)(O'Brien et al., 2004 (link)), rabbit anti-Cx34.1 and mouse anti-Cx35.5 (both generated in this study, 1:100), mouse anti-RMO44 (Life Technologies, 13–0500, 1:100). All secondary antibodies were raised in goat (Life Technologies, conjugated with Alexa-405,–488, −555,–594, or −633 fluorophores, 1:250). Neurobiotin and Biotin Dextran were detected using fluorescently-tagged streptavidin (Life Technologies, conjugated with Alexa-633 fluorophores, 1:500).
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