All sections were deparaffinized in xylene and rehydrated through an ethanol gradient. Sections were then incubated with primary antibodies at 4°C overnight. After rinsing, sections were incubated with Alexa 488‐conjugated goat anti‐mouse IgG and Alexa 546‐conjugated goat anti‐rabbit IgG (Invitrogen, Carlsbad, CA), and then counterstained with DAPI. Images were captured using a confocal laser microscope system (Nikon A1, Nikon, Tokyo, Japan).
Alexa 546 conjugated goat anti rabbit igg
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa 546-conjugated goat anti-rabbit IgG is a fluorescently labeled secondary antibody. It is designed to detect and bind to rabbit primary antibodies, allowing for visualization and detection in various immunological applications.
Automatically generated - may contain errors
Lab products found in correlation
Alexa 488 conjugated goat anti mouse igg, by Thermo Fisher Scientific (3 mentions)
Alexa 488 conjugated goat anti rabbit igg, by Thermo Fisher Scientific (3 mentions)
Alexa 546 conjugated goat anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Rabbit anti cleaved caspase 3 asp175, by Cell Signaling Technology (2 mentions)
Goat serum, by GE Healthcare (2 mentions)
Alexa 647 conjugated goat anti rat igg, by Thermo Fisher Scientific (2 mentions)
Fv10 asw 4.2 viewer image software, by Olympus (2 mentions)
Rabbit anti gaba, by Merck Group (1 mentions)
Alexa 647 conjugated goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Lsm 510 meta confocal microscope, by Zeiss (1 mentions)
Rabbit polyclonal anti gfp, by Thermo Fisher Scientific (1 mentions)
Rabbit anti 5 ht, by Merck Group (1 mentions)
Chicken anti gfp, by Abcam (1 mentions)
Alexa 488 conjugated goat anti mouse immunoglobulin g, by Thermo Fisher Scientific (1 mentions)
Lsm 780, by Zeiss (1 mentions)
Vectastain abc kit, by Vector Laboratories (1 mentions)
Eclipse e800, by Nikon (1 mentions)
Mab5280, by Merck Group (1 mentions)
Ba 1000, by Vector Laboratories (1 mentions)
Vb 6010, by Keyence (1 mentions)
14 protocols using alexa 546 conjugated goat anti rabbit igg
1
Immunofluorescence Imaging of Tissue Samples
2
Immunostaining of Drosophila CNS
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The CNS was dissected from flies in PBS, and fixed in 4% paraformaldehyde in PBS for 60 min. Immunostaining was carried out as described previously16 (link), using the following antibodies at the indicated dilutions: rabbit anti-GABA diluted at 1:500 (Sigma), rabbit anti-5-HT at 1:500 (Sigma), chicken anti-GFP at 1:1,000 (Abcam), rabbit polyclonal anti-GFP at 1:1,000 (Molecular Probes) and mouse monoclonal nc82 at 1:20 (DSHB, University of Iowa, IA). The secondary antibodies used were as follows: Alexa647-conjugated goat anti-mouse IgG at 1:200, Alexa546-conjugated goat anti-rabbit IgG at 1:200, Alexa488-conjugated goat anti-chicken IgG at 1:200, Alexa488-conjugated goat anti-rabbit IgG at 1:200 and Alexa546-conjugated goat anti-mouse IgG at 1:200 (all from Invitrogen). Stacks of optical sections were obtained with a Zeiss LSM 510 META confocal microscope and were processed with Image J software.
3
Dual Immunofluorescence of Nogo-A, Cx47, Cx32, and SOD1 in Paraffin Sections
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Using the same set of paraffin sections, double immunofluorescence staining was performed with the following combinations of antibodies: rabbit polyclonal anti-Nogo-A and mouse monoclonal anti-Cx47; rabbit polyclonal anti-Nogo-A and mouse monoclonal anti-Cx32; rabbit polyclonal anti-SOD1 and mouse monoclonal anti-Cx47. All sections were deparaffinized in xylene and rehydrated through an ethanol gradient. Sections were then incubated with primary antibodies overnight at 4°C. After rinsing, sections were incubated with an Alexa 488-conjugated goat anti-mouse immunoglobulin G (IgG) and an Alexa 546-conjugated goat anti-rabbit IgG (Invitrogen), and then counterstained with 4',6-diamidino-2-phenylindole (DAPI). Images were captured using a confocal laser microscope system (Nikon A1, Nikon, Tokyo, Japan). We used the sequential multiple fluorescence scanning mode to avoid non-specific overlap of colors, and captured all pictures under the same conditions of magnification, laser intensity, gain and offset values, and pinhole setting.
4
Immunofluorescence Staining Protocol
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Sections were deparaffinized through xylene and ethanol. They were then autoclaved in 10 mM citrate buffer at 120 °C for 10 min. After cooling, the sections were blocked in 5% normal goat serum in PBS with 0.5% Triton X-100 before being incubated with primary antibodies overnight at 4 °C. Details of the primary antibodies are provided in Supplementary Table 1. The next day, the sections were washed before being incubated with Alexa 488‐conjugated goat anti‐mouse IgG and Alexa 546‐conjugated goat anti‐rabbit IgG (Invitrogen, Waltham, MA, USA) secondary antibodies for 2 h. 4′,6-diamidino-2-phenylindole (DAPI) was used for nuclear staining. Fluorescent images were obtained using a confocal laser microscope system (Nikon A1; Nikon, Tokyo, Japan). During image acquisition, consistent microscopic settings were implemented for all images within each experimental set, and images were captured from corresponding regions. We used sequential scanning to minimize the effects of crosstalk, and used line averaging and a slow scan speed.
5
Histochemical Analysis of Adrenal Gland
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Under deep anesthesia, mice were transcardially perfused with 4% paraformaldehyde. Frozen tissue sections were stained with rabbit anti-TH antibody (1:1000; made in our laboratory) and biotinylated secondary antibody (1:2000; BA-1000; Vector Laboratories). Immunoreactivity was visualized with avidin-biotinylated peroxidase complex (Vectastain ABC kit; Vector Laboratories). Images were captured by a microscope (Eclipse E800; Nikon) with a cooled charge-coupled device camera (VB-6010; Keyence). Slices of the adrenal gland were incubated with 5% normal swine serum followed by the following primary antibodies: mouse anti-TH antibody (1:1000; MAB5280; Merck Millipore) and rabbit anti-AADC antiserum (1:2000; made in our laboratory). The following secondary antibodies were used: Alexa 546-conjugated goat anti-rabbit IgG (1:500; A11010; Invitrogen) and Alexa 488-conjugated goat antimouse IgG (1:500; A11029; Invitrogen). Images were acquired using a confocal microscope (LSM780; Carl Zeiss). Z-stack images were acquired and projected to one image using the “Z project” function with the “Max Intensity” algorithm in FIJI.
6
Immunofluorescence Staining of Tissue Sections
7
Immunohistochemical Analysis of TcCHT7 Localization
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To analyze localization of TcCHT7 protein, cryosections (~12 μm) of pharate adults (5 d-old pupae) treated with dsTcCHT7 or dsTcVer were prepared. Immunohistochemical analysis was carried out as described previously [35 (link)]. Sections were washed with PBST (0.01 M phosphate buffered saline, pH 7.4 containing 0.1% Tween 20) three times, and then blocked with blocking buffer (2% bovine serum albumin in PBST) for 1 h at room temperature. The sections were incubated with anti-TcCHT7 antibody (1:50 in blocking buffer) for 3 h at room temperature, washed with PBST three times and then Alexa 546-conjugated goat anti-rabbit IgG (Invitrogen) secondary antibody (1:300 in blocking buffer) was applied for 1 h at room temperature. After washing the sections with PBST, cuticular chitin and nuclei were stained with fluorescein isothiocyanate (FITC)-conjugated chitin-binding protein probe and TO-PRO-3 (Invitrogen), respectively. Tissues were observed using the LSM700 laser scanning confocal microscope (Zeiss) with appropriate filters.
8
Immunofluorescence Analysis of Tight Junction Proteins
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For immunofluorescence analysis, the following primary antibodies were used: rabbit anti‐ZO‐1 (1:100 dilution, Thermo Fisher, #61‐7300), rabbit anti‐claudin‐5 (1:400, Abcam, #ab53765), rabbit anti‐GFAP (1:500, DAKO, #Z0334), rat anti‐CD31 (clone Mec13.3, BD Biosciences), Pals1 (1:100, Merck, #7‐708), rat anti‐Crb3 (14F9, Abcam, ab180835), mouse anti‐E‐cadherin (1:200, BD Bioscience, #610181), and rabbit anti‐cleaved caspase‐3 (Asp175, 1:100 Cell Signaling, #9661). Secondary antibodies were 3 mg/ml Alexa 546‐conjugated goat anti‐rabbit IgG and 3 mg/ml Alexa 488‐conjugated goat anti‐rat IgG (all from Invitrogen). For Western blotting, rabbit anti‐MPDZ (1:500, Thermo Scientific, 42‐2700) and mouse anti‐beta actin (1:2,500, Sigma‐Aldrich #A5441) were used.
9
Immunostaining of ADAM10 and HLA Class I
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Cell were fixed with 4% paraformaldehyde and either permeabilized with 0.5% saponin or used as they were for immunostaining with goat anti-ADAM10 (AB936, R&D-Systems), rabbit anti-ADAM10 pro-domain (ab39178, Abcam), mouse anti-HLA class I (Santa-Cruz Biotechnology), mouse anti-ß-actin (Sigma) and IgGs purified from Crc patient sera (50 μg/ml). Alexa-488-conjugated goat-anti-rabbit IgG, donkey-anti-goat IgG and rabbit-anti-mouse IgG (Invitrogen) or FITC-conjugated goat-anti-human IgG (Southern Biotechnology) were used as secondary Abs; in double staining experiments the Alexa-546-conjugated goat-anti-rabbit IgG (Invitrogen) was used. Staining was assessed either by immunofluorescence microscopy (Zeiss Upright Axo Imager 2) or confocal microscopy (Leica TCS SP5 Laser Scanning Confocal), and images acquired with AxoVision Rel.4.8.2 (Zeiss) or LAS-AF (Leica) software, respectively. Binding-competition was performed by pre-incubating cells (7 hr at 4°C) with 50 μg/ml of either rabbit-anti-ADAM10 pro-domain (ab39178, Abcam) or purified rabbit IgG before immunofluorescence staining with IgG purified from patient sera.
10
Multicolor Immunostaining of Cryosections
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Cryosections (10 μm) on Superfrost Plus slides were equilibrated at RT, fixed in cold methanol, washed in PBS and blocked in PBS containing 0.05% Tween-20, 5% FBS, 5% BSA, and 5% goat serum (GE Healthcare, UK) for 1 h. The sections were immunostained with rabbit anti-fluorescein (cat #A889, Thermo Fisher Scientific, MA, USA), rat anti-mouse CD31 (cat #553371, BD Pharmingen, USA), rat anti-mouse CD68 (cat #MCA1957GA, AbD Serotec, USA), rat anti-mouse CD206 (cat #MCA2235GA, Bio-Rad, USA), rat antimouse LYVE-1 (cat #14-0443, Affymetrix, USA), and rabbit anti-cleaved caspase-3 (cat #966, Cell Signaling Technology, USA) as primary antibodies. Alexa 488-conjugated goat anti-rabbit IgG, Alexa 647conjugated goat anti-rat IgG, Alexa 546-conjugated goat anti-rabbit IgG (all Invitrogen, Thermo Fisher Scientific, USA) were used as secondary antibodies. To detect endogenous IgG as a marker of blood vessel leakiness, we stained to tissue sections with Alexa 546 goat anti-mouse IgG, (#Cat.No: A11003; Invitrogen, Thermo Fisher Scientific, USA) at 1/400 dilution. Nuclei were counterstained with 1 μg/ml DAPI. The tissue sections were examined by Olympus FV1200MPE confocal microscope (Olympus, Germany), and the images were processed and analyzed using the FV10-ASW 4.2 Viewer image software (Olympus, Germany) and Image J freeware.
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