BV2 microglial cells were grown on poly-L-lysine coated coverslips. Following transfection and/or treatment, cells were fixed with 4% paraformaldehyde for 15min at room temperature. Permeabilization of cell membranes was achieved using 0.1% Triton-X containing PBS. Following this, the slides were blocked using 5% normal goat serum and incubated overnight at 4°C with the following antibodies- SMAD4-1:100 (Santa Cruz, sc-7966), pSMAD2/3-1:200 (Cell signaling technology, Cat No: 8828) and SMAD2/3-1:200 (Cell Signaling Technology, Cat No: 8685). Following this, cells were incubated with fluorophore tagged secondary antibodies: anti-rabbit Cy3 (Sigma, Cat No: c2306), anti-mouse Cy3 (Sigma, Cat No: C2181) and FITC conjugated lectin (Sigma, Cat No: L0401) was used as a marker for microglial cells. Cell nuclei were counterstained with DAPI for visualization. Fluorescence images were captured using a confocal microscope (Olympus, FV1000 Fluoview).
Anti rabbit cy3
Manufactured by Merck Group
Sourced in Canada
Anti-rabbit Cy3 is a fluorescent dye conjugate used for labeling and detecting rabbit-derived antibodies in various biological assays. It emits light in the red-orange region of the visible spectrum upon excitation, allowing for sensitive visualization of target proteins or cells.
Automatically generated - may contain errors
Lab products found in correlation
Metamorph software, by Universal Imaging (4 mentions)
Anti mouse oregon green, by Thermo Fisher Scientific (3 mentions)
Anti mouse cy3, by Merck Group (2 mentions)
Dmire2 microscope, by Leica (2 mentions)
Orca charge coupled device camera, by Hamamatsu Photonics (2 mentions)
Mouse anti ha epitope igg, by Santa Cruz Biotechnology (2 mentions)
Mouse anti myc 9e10, by Merck Group (2 mentions)
Orca ccd camera, by Hamamatsu Photonics (2 mentions)
Fluoview fv1000, by Olympus (1 mentions)
Fitc conjugated lectin, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Rabbit anti gfp, by Thermo Fisher Scientific (1 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
Alexa fluor 594 anti rabbit, by Thermo Fisher Scientific (1 mentions)
Axioskop 2 plus, by Zeiss (1 mentions)
Brn3a, by Santa Cruz Biotechnology (1 mentions)
Phospho s6 ser240 244, by Cell Signaling Technology (1 mentions)
Calbindin, by Swant (1 mentions)
Mouse anti gfp, by Merck Group (1 mentions)
Anti mouse alexa 488, by Thermo Fisher Scientific (1 mentions)
10 protocols using anti rabbit cy3
1
Immunocytochemical Analysis of Microglial Signaling Pathways
2
Immunofluorescence Analysis of Cellular Components
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Immunofluorescence analysis (IFA) was as described 64 (link). Antibodies for IFA were used at the following
dilutions: mouse anti-HA-epitope IgG (Santa Cruz Biotechnology Inc.) at 1:1000,
rabbit anti-Rab11 at 1:200 44 (link), rabbit
anti-Rab5a at 1:200 68 (link), mouse anti-p67 at
1:1000 43 (link), mouse anti-BiP at 1:10 000
41 (link), rabbit anti-VSG221 at 1:1000
68 (link), rabbit anti-RabX2 at 1:50 71 (link), rabbit anti-IGP48 (this study) at 1:50.
Secondary antibodies were used at the following dilutions: anti-mouse Oregon
Green (Molecular Probes) at 1:1000 and anti-rabbit Cy3 (Sigma) at 1:1000. Cells
were examined on a Nikon Eclipse E600 epifluorescence microscope fitted with
optically matched filter blocks and a Hamamatsu ORCA charge-coupled-device
camera. Digital images were captured using Metamorph software (Universal Imaging
Corp.) on a computer running the Windows XP operating system (Microsoft Inc.)
and the raw images processed using Photoshop CS6 software (Adobe Systems Inc.).
Confocal z-sections were acquired using a Leica DMIRE2 microscope and
deconvolved using Huygens Professional software.
dilutions: mouse anti-HA-epitope IgG (Santa Cruz Biotechnology Inc.) at 1:1000,
rabbit anti-Rab11 at 1:200 44 (link), rabbit
anti-Rab5a at 1:200 68 (link), mouse anti-p67 at
1:1000 43 (link), mouse anti-BiP at 1:10 000
41 (link), rabbit anti-VSG221 at 1:1000
68 (link), rabbit anti-RabX2 at 1:50 71 (link), rabbit anti-IGP48 (this study) at 1:50.
Secondary antibodies were used at the following dilutions: anti-mouse Oregon
Green (Molecular Probes) at 1:1000 and anti-rabbit Cy3 (Sigma) at 1:1000. Cells
were examined on a Nikon Eclipse E600 epifluorescence microscope fitted with
optically matched filter blocks and a Hamamatsu ORCA charge-coupled-device
camera. Digital images were captured using Metamorph software (Universal Imaging
Corp.) on a computer running the Windows XP operating system (Microsoft Inc.)
and the raw images processed using Photoshop CS6 software (Adobe Systems Inc.).
Confocal z-sections were acquired using a Leica DMIRE2 microscope and
deconvolved using Huygens Professional software.
3
Immunofluorescence Microscopy Techniques
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Samples for IF were prepared as previously described (Leung et al., 2008). Antibodies were used at the following dilutions: mouse and rabbit anti-HA epitope IgG (both from Santa Cruz Biotechnology Inc.) at 1:1000, mouse 9E10 anti-myc (Sigma) at 1:1000, rabbit anti-GFP (Life Technologies) at 1:500, rabbit anti-ISG65 and rabbit anti-ISG75 (from P. Overath, Tubingen) at 1:1000, rabbit anti-CatL at 1:1000 (from J. Bangs, Buffalo), mouse anti-GLP-1 at 1:1000 (from D. Russell, Cornell). Secondary antibodies were used at the following dilutions: anti-rabbit Cy3 (Sigma) at 1:1000. Coverslips were mounted using Vectashield mounting medium supplemented with 4’,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Inc.). The cells were examined on a Nikon Eclipse E600 epifluorescence microscope fitted with optically matched filter blocks and a Hamamatsu ORCA CCD camera. Digital Images were captured using Metamorph software (Universal Imaging Corp.) on a Windows XP computer (Microsoft Inc.), and the raw images were processed using Adobe Photoshop CS3 (Adobe Systems Inc.).
4
Immunofluorescence Analysis of Cellular Compartments
5
Immunoblotting and Microscopy Protocols
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Samples were prepared as described previously (87 (link)). Antibodies were used at the following dilutions: mouse and rabbit anti-HA epitope IgG (sc-57594 and sc-7392, Santa Cruz Biotechnology) at 1:1000, mouse 9E10 anti-Myc at 1:1000 (Sigma), rabbit anti-ISG75 (from P. Overath) at 1:1000, rabbit anti-CatL at 1:1000 (85 (link)), and mouse anti-GLP-1 at 1:1000 (86 (link)). Rabbit anti-PFK (Do405, 1:1000), rabbit anti-PGK (Do425, 1:1000), and rabbit anti-PYK (SB953, 1:500) were all from P. Michels. Secondary antibodies were used at the following dilutions: anti-mouse Oregon Green (Molecular Probes) at 1:1000 and anti-rabbit Cy3 (Sigma) at 1:1000. Cells were examined on a Nikon Eclipse E600 epifluorescence microscope fitted with optically matched filter blocks and a Hamamatsu ORCA CCD camera. Digital images were captured using Metamorph software (Universal Imaging Corp.), and raw images were processed using Adobe Photoshop CS3 (Adobe Systems Inc.).
6
Retinal Cryosectioning and Immunohistochemistry
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Animals were perfused transcardially with 4% PFA and retinal
cryosections (16 μm) were prepared as previously
described by us.67 (link) Some
cryosections were prepared from eyes labeled with the retrograde tracer
FG (Fluorochrome, Englewood, CO, USA), which was applied to the superior
colliculus 1 week before optic nerve axotomy as described by
us.66 (link) Each of the
following primary antibodies were added to the retinal sections in
blocking solution (3% bovine serum albumin, 0.3% Triton
X-100) and incubated overnight at 4 °C: phospho-S6 (Ser
240/244, 1 : 200; Cell Signaling Technology, Boston,
MA, USA), βIII-tubulin (TUJ1, 1 : 400;
Sigma-Aldrich), calbindin (1 : 200; Swant, Marly,
Switzerland), REDD2 (5 μg/ml; Biorbyt, San
Francisco, CA, USA), REDD1 (1 μg/ml; ProSci Inc.,
Poway, CA, USA) or Brn3a (1 μg/ml; Santa Cruz
Biotechnologies). The secondary antibodies used were as follows:
anti-rabbit Cy3 (1.5 μg/ml; Sigma-Aldrich),
anti-mouse FITC (1 : 1000; Sigma-Aldrich), anti-rabbit
Alexa Fluor 594 (2 μg/ml; Molecular Probes) or
anti-goat Alexa Fluor 488 (2 μg/ml; Molecular
Probes). Fluorescent labeling was observed with a Zeiss AxioSkop 2 Plus
(Carl Zeiss Canada).
cryosections (16 μm) were prepared as previously
described by us.67 (link) Some
cryosections were prepared from eyes labeled with the retrograde tracer
FG (Fluorochrome, Englewood, CO, USA), which was applied to the superior
colliculus 1 week before optic nerve axotomy as described by
us.66 (link) Each of the
following primary antibodies were added to the retinal sections in
blocking solution (3% bovine serum albumin, 0.3% Triton
X-100) and incubated overnight at 4 °C: phospho-S6 (Ser
240/244, 1 : 200; Cell Signaling Technology, Boston,
MA, USA), βIII-tubulin (TUJ1, 1 : 400;
Sigma-Aldrich), calbindin (1 : 200; Swant, Marly,
Switzerland), REDD2 (5 μg/ml; Biorbyt, San
Francisco, CA, USA), REDD1 (1 μg/ml; ProSci Inc.,
Poway, CA, USA) or Brn3a (1 μg/ml; Santa Cruz
Biotechnologies). The secondary antibodies used were as follows:
anti-rabbit Cy3 (1.5 μg/ml; Sigma-Aldrich),
anti-mouse FITC (1 : 1000; Sigma-Aldrich), anti-rabbit
Alexa Fluor 594 (2 μg/ml; Molecular Probes) or
anti-goat Alexa Fluor 488 (2 μg/ml; Molecular
Probes). Fluorescent labeling was observed with a Zeiss AxioSkop 2 Plus
(Carl Zeiss Canada).
7
Immunolocalization of PIN2 and GFP
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Three‐ to 4‐d‐old seedlings were immunolocalized using an in situ pro robot (Intavis, Cologne, Germany) according to the described protocol (Sauer et al., 2006b ). The primary antibodies were rabbit anti‐PIN2 (Abas et al., 2006 ) 1 : 1000 and mouse anti‐GFP (Sigma‐Aldrich) 1 : 600, and the secondary antibodies were Cy3 anti‐rabbit (Sigma‐Aldrich) 1 : 600 and Alexa488 anti‐mouse (Invitrogen) 1 : 600.
8
Antibody Panel for WB and IF
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For Western blot (WB) and immnufluorescence, we employed: rabbit anti-LRP1β (Sigma–Aldrich Canada), rabbit anti-LC3B (Sigma–Aldrich), mouse anti-β-actin (GenScript), mouse anti-GM130 (Sigma), anti-rabbit HRP (Sigma), mouse anti-HRP (Sigma), Alexa Flour 488 anti-rabbit (Sigma), Cy3 anti-rabbit (Sigma), Cy3 anti-mouse, and Alexa Flour 594 anti-rabbit (Sigma).
9
Immunolocalization of ARF1 in Arabidopsis
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For immunolocalization assay, 4-day-old seedlings were fixed with 4% paraformaldehyde (Merck) using vacuum infiltration for 60 min at room temperature. Automated whole-mount protein immunolocalization was performed as described previously (Sauer et al., 2006 (link)). The anti-ARF1 rabbit antibody (Pimpl et al., 2000 (link)) was used at 1:600 dilution. For the secondary antibody, we used CY3 anti-rabbit (Sigma-Aldrich) at a 1:600 dilution.
10
Antibody-based Protein Detection in Endothelial Cells
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The expressions of total eNOS and β-actin were detected with monoclonal antibodies (Invitrogen and BD Biosciences, resp.), while PSer1179eNOS and Caveolin 1 (Cav1) were evidenced with polyclonal antibodies that were purchased, respectively, from Invitrogen and Sigma. heparan sulphate was stained with a mouse monoclonal antibody (MAB2040, Millipore) that we labeled with Alexa Fluor 488 using the APEX Antibody Labeling Kit (Invitrogen).
The secondary antibodies employed for immunofluorescence experiments were Alexa Fluor 488 anti-mouse (Molecular Probes) for total eNOS and Cy3 anti-rabbit (Sigma) for PSer1179eNOS and Cav1. For Western blot experiments we used horseradish peroxidase-conjugated secondary antibodies: anti-mouse for β-actin (Invitrogen) and anti-rabbit for PSer1179eNOS (Amersham).
The secondary antibodies employed for immunofluorescence experiments were Alexa Fluor 488 anti-mouse (Molecular Probes) for total eNOS and Cy3 anti-rabbit (Sigma) for PSer1179eNOS and Cav1. For Western blot experiments we used horseradish peroxidase-conjugated secondary antibodies: anti-mouse for β-actin (Invitrogen) and anti-rabbit for PSer1179eNOS (Amersham).
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