Immunofluorescent staining was performed on 8-μm thick sections of the cryopreserved tissue samples, as previously described.27 (link) Briefly, the tissue sections were fixed in 2% formaldehyde, and the staining was done sequentially. The following primary monoclonal antibodies were used; rabbit anti-human CD4 antibody (clone EPR6855; Abcam, Cambridge, England), mouse anti-human CCR5 antibody (clone MC-5, kindly provided by Professor M. Mack from the University Clinic of Regensburg, Germany), and rat anti-human Langerin antibody (clone 929F3.01; Dendritics, Lyon, France). Fluorescent-labeled secondary antibodies used were Alexa Fluor 594-conjugated donkey anti-rabbit IgG antibody, Alexa Fluor 488-conjugated donkey anti-mouse IgG antibody, and Alexa Fluor 488-conjugated donkey anti-rat IgG antibody (Molecular Probes, Life Technologies Europe BV, Stockholm, Sweden). Antigen retrieval (pure methanol +0.5% hydrogen peroxide) was needed before addition of the primary CD4 antibody. Tissue sections were counterstained with DAPI (Molecular Probes, Life Technologies) to stain the nuclei, washed and thereafter mounted with Fluorescent Mounting Medium (Dako, Carpinteria, CA). Negative controls were included and consisted of incubations in the presence of secondary antibodies alone.
Alexafluor 594 conjugated donkey anti rabbit igg antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
AlexaFluor 594-conjugated donkey anti-rabbit IgG antibody is a secondary antibody used to detect and visualize rabbit primary antibodies in various immunoassays and imaging applications. The antibody is conjugated with the AlexaFluor 594 fluorescent dye, which emits red fluorescence upon excitation.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 conjugated donkey anti mouse igg antibody, by Thermo Fisher Scientific (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Fluorescent mounting medium, by Agilent Technologies (1 mentions)
Bz x710, by Keyence (1 mentions)
Dapi 4 6 diamidino 2 phenylindole, by Thermo Fisher Scientific (1 mentions)
Bx50 bx fla, by Olympus (1 mentions)
Fluoromount, by Diagnostic BioSystems (1 mentions)
Alexa fluor 488 conjugated donkey anti goat igg antibody, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Anti hp1α, by Cell Signaling Technology (1 mentions)
Hpa021638, by Merck Group (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Dmi8 microscope, by Leica (1 mentions)
Nucblue live readyprobes reagent, by Thermo Fisher Scientific (1 mentions)
Application suite x las x, by Leica (1 mentions)
7 protocols using alexafluor 594 conjugated donkey anti rabbit igg antibody
1
Immunofluorescent Staining of Cryopreserved Tissue
2
Immunofluorescent Labeling of Testicular Structures
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Sections of PFA-fixed, paraffin-embedded testes, after deparaffinization and rehydration, were immersed in 20 mM Tris-HCl buffer (pH 9.0) and heated at 95°C for 15 min using the autoclave apparatus to retrieve antigens (Nakata et al. 2015a) . After cooling to room temperature, sections were washed in PBS and treated with 1% skim milk/PBS at room temperature for 1 h to prevent non-specific antibody binding. They were then incubated with a rabbit polyclonal anti-laminin antibody (L9393; Sigma-Aldrich) at a concentration of 7.5 μg/ mL in PBS at 4°C overnight. After washing in PBS, sections were incubated with an Alexa Fluor 594-conjugated donkey anti-rabbit IgG antibody (Molecular Probes) at a 1:400 dilution at room temperature for 1 h. Sections of P21 testes were further incubated with the Alexa Fluor 488 conjugate of peanut agglutinin (PNA) (Molecular Probes), a marker of the acrosome (Nakata et al. 2015a (Nakata et al. , 2017)) (link), at a 1:400 dilution at room temperature for 30 min. All sections were counterstained in the nuclei with DAPI (4′6-diamidino-2-phenylindole; Molecular Probes) at 300 nM, mounted on glass slides with Fluoromount (Diagnostic BioSystems, Pleasanton, CA, USA) and examined with a fluorescence microscope (BX50/BX-FLA; Olympus, Tokyo, Japan, or and BZ-X710; Keyence, Osaka, Japan).
3
Double Immunofluorescent Labeling of Phospho-CREB
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Double immunofluorescent labeling was performed as follows: (1) sections were dried 30 min at room temperature and washed with Tris-buffered saline; (2) sections were blocked 1 h in Tris-buffered saline containing 10% normal horse serum (NHS) and 0.3% Triton X-100 (TX-100) at room temperature; (3) sections were incubated overnight at 4°C in Tris-buffered saline/1% NHS/0.3% TX-100 containing a mixture of a goat anti-c-FOS antibody (sc-52, diluted 1:400; Santa Cruz) and a rabbit anti-phospho-CREB (P-CREB, Ser133) antibody (87G3, diluted 1:100, Cell Signaling); (4) sections were rinsed in Tris-buffered saline and incubated 1 h at room temperature in Tris-buffered saline /1% NHS/0.3% TX-100 containing a mixture of Alexa Fluor 488 conjugated donkey anti-goat IgG antibody (1:300) and Alexa Fluor 594 conjugated donkey anti-rabbit IgG antibody (1:300, Life Technologies); and (5) sections were rinsed in Tris-buffered saline and covered with Prolong with DAPI. Negative immunofluorescent controls were performed by omitting the primary antibody from the otherwise regular immunolabeling protocol.
4
Quantifying Tissue Hypoxia Levels
5
Comprehensive Antibody Panel for Nuclear Protein Analysis
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Rabbit polyclonal anti-RCOR2 (Sigma HPA021638, lot number A117885), anti-coilin antibody (Genetex, GTX112570), anti-H3K9me3 (Abcam, ab8898), anti-HP1α (Cell Signaling, #2616S), anti-LSD1 (Abcam, ab17721), anti-SON (Thermo Fisher, #PA5-65107), anti-SRRM2 (Thermo Fisher, #PA5-66827), anti-HDAC2 (Abcam, #ab7029) and anti-GAPDH (Cell Signaling, #2118S). Rabbit monoclonal anti-HA (Cell Signaling, #3724S). We used anti-SC35 mouse monoclonal antibodies from (Genetex GTX11826) and (Sigma SAB4200725). Recently, it was shown that this monoclonal antibody recognizes the SRRM2 and SRSF7 proteins but does not recognize SC35 [14 (link)]. Mouse monoclonal anti-nucleophosmin (Abcam, ab10530), anti-SRSF1/ASF-1 (Millipore-Sigma, #MABE936). Alexa Fluor® 488 AffiniPure Fab Fragment Goat Anti-Rabbit IgG (H + L) (Jackson Immunoresearch, 111-547-003). AlexaFluor 594-conjugated donkey anti-rabbit IgG antibody (Invitrogen, Thermo Fisher, R37119). AlexaFluor 488-conjugated donkey anti-mouse IgG antibody (Invitrogen, Thermo Fisher, R37114).
6
Immunofluorescence Staining of Cultured Cells
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The IF protocol was performed as described previously [20 (link)]. Cultured cancer cells were rinsed with phosphate-buffered saline (PBS) three times and fixed with 3.7% formaldehyde. Then the cells were permeabilized with 0.1% Triton X-100. After 1 h of blocking with 1% bovine serum albumin, the cells were then incubated with a primary antibody overnight. Then the cells were washed and incubated (in the dark) with an Alexa Fluor 488- or Alexa Fluor 594-conjugated donkey anti-rabbit IgG antibody (Invitrogen, Grand Island, NY) for 1 h at room temperature. The cells were washed with PBS (containing 0.02% Tween 20), stained by mounting on a slide with aqueous mounting medium containing 0.5 mg/mL 4′,6-diamidino-2-phenylindole and examined with a fluorescence microscope.
7
Immunofluorescent Staining of DENV NS1 and Calnexin
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Cells were fixed with 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 15 min at RT and then permeabilised with 0.5% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) in PBS for 10 min at RT. Cells were blocked with 2.5% normal horse serum (NHS; Cytiva, Marlborough, MA, USA) in PBS for 30 min at RT, followed by staining with mouse monoclonal anti-DENV NS1 antibody (1:1000; clone FE8, The Native Antigen Company, Oxford, UK) and rabbit polyclonal anti-calnexin antibody (1:500; Sigma-Aldrich, St. Louis, MO, USA) for 1 h at RT. Afterwards, Alexa Fluor 488–conjugated donkey anti-mouse IgG antibody (1:500; Invitrogen, Waltham, MA, USA) and Alexa Fluor 594–conjugated donkey anti-rabbit IgG antibody (1:500; Invitrogen, Waltham, MA, USA) along with NucBlue Live ReadyProbes Reagent (Hoechst 33342, Invitrogen, Waltham, MA, USA) were added for 1 h at RT in the dark. All antibodies were diluted in 2.5% NHS/PBS and cells were washed three times with PBS after each antibody incubation. Images were captured using a Leica DMi8 microscope coupled to a Leica DFC3000 G camera and processed using Leica Application Suite X (LAS X; all Leica Microsystems, Wetzlar, Germany).
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