To explore the colocalisation of β5i and RIG-I, double-label immunostaining was performed. After blocking with 5% bovine serum albumin in PBST at room temperature for 1 hour, the samples were treated with mouse anti-RIG-I monoclonal antibody (1:50, sc-376845, Santa Cruz Biotechnology) and rabbit anti-β5i monoclonal antibody (1:2000, ab180606, Abcam) overnight at 4°C, followed by three washes with PBS (10 min each wash). Next, the samples were treated with Alexa Fluor 594-conjugated goat anti-mouse IgG (1:200, 8890, Cell Signaling Technology) and Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:200, 4412, Cell Signaling Technology), which were used as the secondary antibodies, in a dark chamber for 1 hour, followed by three washes with PBST. After counterstaining with DAPI (Beyotime, Shanghai, China), the slides were mounted with coverslips. To clarify whether β5i is expressed in regenerative myocytes, we performed double-label immunostaining using rabbit anti-β5i monoclonal antibody (1:2000, ab180606, Abcam) and mouse anti-NCAM1 monoclonal antibody (1:500, ab6123, Abcam), following the same procedure mentioned above. In addition, double-label immunofluorescence staining using rabbit anti-β5i monoclonal antibody and mouse anti-MxA monoclonal antibody (1:100, sc-166412, Santa Cruz) was performed following the above procedure.
Alexa fluor 594 conjugated goat anti mouse igg
Manufactured by Cell Signaling Technology
Sourced in United States
Alexa Fluor 594-conjugated goat anti-mouse IgG is a secondary antibody that binds to mouse immunoglobulin G (IgG). The antibody is conjugated with Alexa Fluor 594, a fluorescent dye that can be detected using appropriate instrumentation.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 conjugated goat anti rabbit igg, by Cell Signaling Technology (3 mentions)
Dm5000b, by Leica (3 mentions)
Ab9829, by Abcam (2 mentions)
Cd163, by Cell Signaling Technology (2 mentions)
Cd206, by Cell Signaling Technology (2 mentions)
Ab180606, by Abcam (1 mentions)
Ab6123, by Abcam (1 mentions)
Sc 376845, by Santa Cruz Biotechnology (1 mentions)
Sc 166412, by Santa Cruz Biotechnology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
High performance liquid chromatography (hplc), by Chengdu Must Bio-Technology (1 mentions)
Ab18256, by Abcam (1 mentions)
A110694, by Sangon (1 mentions)
E607303, by Sangon (1 mentions)
E672002, by Sangon (1 mentions)
Sc 17796, by Santa Cruz Biotechnology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Sangon (1 mentions)
Axio observer inverted fluorescence microscope, by Zeiss (1 mentions)
Prolong diamond antifade mountant with dapi, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 555 conjugated goat anti rabbit igg, by Cell Signaling Technology (1 mentions)
8 protocols using alexa fluor 594 conjugated goat anti mouse igg
1
Colocalization of β5i and RIG-I
2
Visualizing SARS-CoV-2 Spike Protein Localization
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Confocal microscopy analysis was performed to study the localization of the S protein. Briefly, 293 T cells overexpressing SFL, Sdel18, SG614, SFko, and 293 T-ACE2-GFP were cocultured in 24-well plates for 12 h, and SARS-CoV-2 S proteins were detected using monoclonal mouse anti-SARS Spike glycoprotein antibody 1A9 (1:500). Specific signals were visualized using Alexa Fluor 594-conjugated goat anti-mouse IgG (1:500) (Cell Signaling Technology, USA). For nuclear staining, cells were treated with DAPI for 5 min. Staining sections were analyzed using a laser scanning confocal microscope (Zeiss 880).
3
Immunohistochemical Analysis of PCa Biomarkers
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Assays were performed as previously reported [20 (link)]. In brief, for the IF experiment, cells were incubated with primary antibodies against CCL20 (1:200, #ab9829) (Abcam) at 4 °C overnight, then incubated with the fluorescent secondary antibody Alexa Fluor 594-conjugated goat anti-mouse IgG (Cell Signaling Technology) and imaged using a fluorescence microscope (DM5000B, Leica). For IHC, the paraffin sections were incubated with antibodies against CCL20 (1:200, #ab9829) (Abcam). Intensity scores were recorded as: 0 (no staining), 1 (weakly staining, light yellow), 2 (moderately staining, yellowish brown), and 3 (strongly staining, brown). In addition, we also detected the expression of ki67 (1:1000, #9449), CD68 (1:200, #97778), CD163 (1:400, #93498), CD206 (1:200, #24595) and CD31 (1:100, #77699) (Cell Signaling Technology) in PCa tissue or xenograft tissue. Images were observed under an Olympus multifunction microscope (Olympus BX51, Tokyo, Japan). All evaluations were performed by three independent senior pathologists using the same microscope.
4
GRb1 Analytical Standard and Antibody Evaluation
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The GRb1 analytical standard (CAS No: 41753-43-9; C54H92O23) with ≥98.0% purity (HPLC) was purchased from Chengdu Must Biotechnology Co., Ltd. (Chengdu, China). The chemical structure of GRb1 is presented in Figure 1A . Antibodies against NF-κB (ET1603-12) were obtained from HuaAn Biotechnology Co., Ltd. (Hangzhou, China), and anti-TNF-α (ab1793) antibodies were acquired from Abcam (Cambridge, United Kingdom). Horseradish peroxidase-labeled goat anti-mouse/rabbit IgG (GK500710) was procured from Gene Tech (Shanghai, China). Alexa Fluor594-conjugated goat anti-rabbit IgG (#8889) and Alexa Fluor594-conjugated goat anti-mouse IgG (#8890) were obtained from Cell Signaling Technology (United States).
5
Immunofluorescence Analysis of HMGB1 and BRG1
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Cells were fixed with 4% paraformaldehyde (E672002, Sangon Biotech) for 15 min at room temperature followed by incubation with 0.5% Triton solution (A110694, Sangon Biotech) for 10 min to permeabilize the cell membrane. Then, cells were incubated with Tris-buffered saline containing 5% bovine serum albumin (BSA) for 30 min. Subsequently, samples were incubated with rabbit anti-HMGB1 (#ab18256, Abcam) and mouse anti- BRG1 (#sc17796, Santa Cruz) at 4 ℃ overnight. Finally, the fluorescent secondary antibody Alexa Fluor 488-conjugated goat anti-rabbit IgG (#4412S, Cell Signaling Technology) and Alexa Fluor 594-conjugated goat anti-mouse IgG (#8890, Cell Signaling Technology) were used to detect primary antibodies. DAPI (E607303, Sangon Biotech) was applied for nuclear staining. Fluorescence images were visualized and collected under inverted confocal microscopy (DM5000B, Leica).
6
FRY Expression Immunofluorescence Assay
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Immunofluorescence was used to assess the in-situ expression of the FRY. MDA-MB-231 related cells were cultured on coverslips in DMEM medium with 10% FBS and penicillin/streptomycin at 37°C with 5% CO2 for 24 h. The cells were briefly washed in PBS, fixed with 4% formaldehyde for 30 min, permeabilized with 0.5% Triton X-100 in PBS for 10 min and incubated in blocking buffer (10% goat serum, 0.2% Triton X-100, 0.05% Tween-20 in PBS, pH 7.4) for 1 h at room temperature. The cells were then incubated with primary antibodies diluted in blocking buffer overnight at 4°C. After rinsing, the primary antibodies were detected with Alexa Fluor® 594 conjugated goat anti-mouse IgG (Cell Signaling Technology) or Alexa Fluor® 555 conjugated goat anti-rabbit IgG (Cell Signaling Technology) secondary antibodies diluted at 1:500 in blocking buffer for 1 h at room temperature. Coverslips were mounted using ProLong® Diamond Antifade Mountant with DAPI (Life Technologies) and fluorescence images were visualized and captured with a ZEISS Axio Observer inverted fluorescence microscope (ZEISS, Jena, Germany).
Primary antibodies and dilution used in applications include the following: mouse anti-FRY (2F2-D8-E3-G3) antibody (RayBiotech, 1:100), mouse monoclonal anti-β-Actin antibody (Sigma-Aldrich, 1:2,000).
Primary antibodies and dilution used in applications include the following: mouse anti-FRY (2F2-D8-E3-G3) antibody (RayBiotech, 1:100), mouse monoclonal anti-β-Actin antibody (Sigma-Aldrich, 1:2,000).
7
Immunofluorescence Analysis of Brain Slices
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Briefly, brain slices were preincubated with 0.1% Triton X-100 (v/v) in 0.01 M PBS (pH 7.4) for 15 minutes. After blocking with 10% normal goat serum (Sigma-Aldrich), slides were incubated with primary antibodies as follows: rabbit anti-GFAP, rabbit anti-myelin basic protein (MBP), and rabbit anti-IBA1 (1 : 400; Abcam, Cambridge, MA, USA). After incubated overnight at 4°C and rinsed in 0.01 M PBS (3 × 5 minutes), tissue slices were incubated with Alexa Fluor® 488 conjugated goat anti-rabbit IgG (H + L), F(ab')2 Fragment or Alexa Fluor® 594 conjugated goat anti-mouse IgG (H + L), F(ab')2 Fragment (1 : 1000; Cell Signaling Technology) in 0.01 M PBS for 1 hour at room temperature. If necessary, the sections were counterstained for nuclei with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; 1 : 1000; Roche, Mannheim, Germany), and then slides were mounted in the ProLong® Gold antifade reagent (Thermo Fisher Scientific, USA) prior to imaging. The immunofluorescence intensity was analyzed with Image J, v1.8.0.
8
Immunohistochemical Analysis of PCa Biomarkers
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Assays were performed as previously reported [20 (link)]. In brief, for the IF experiment, cells were incubated with primary antibodies against CCL20 (1:200, #ab9829) (Abcam) at 4 °C overnight, then incubated with the fluorescent secondary antibody Alexa Fluor 594-conjugated goat anti-mouse IgG (Cell Signaling Technology) and imaged using a fluorescence microscope (DM5000B, Leica). For IHC, the paraffin sections were incubated with antibodies against CCL20 (1:200, #ab9829) (Abcam). Intensity scores were recorded as: 0 (no staining), 1 (weakly staining, light yellow), 2 (moderately staining, yellowish brown), and 3 (strongly staining, brown). In addition, we also detected the expression of ki67 (1:1000, #9449), CD68 (1:200, #97778), CD163 (1:400, #93498), CD206 (1:200, #24595) and CD31 (1:100, #77699) (Cell Signaling Technology) in PCa tissue or xenograft tissue. Images were observed under an Olympus multifunction microscope (Olympus BX51, Tokyo, Japan). All evaluations were performed by three independent senior pathologists using the same microscope.
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