Sections were dried at room temperature and incubated for 25 min in 10 mM sodium citrate buffer (pH 6.0) at 95°C. For BrdU staining, sections were treated with 2 N HCl for 30 min at room temperature. Sections were then blocked in 3% normal sheep serum and 0.1% Triton X-100 in PBS (blocking buffer) for 1 hour at room temperature. Then, sections were incubated in primary antibodies [rat anti-CTIP2 (1:500; Abcam, ab18465), mouse anti-SATB2 (1:500; Abcam, ab51502), rabbit anti-CUX1 (1:100; Santa Cruz Biotechnology, sc-13024), mouse anti-FOXP2 (1:250; Sigma-Aldrich, AMAB91361), rabbit anti-H3K36me3 (1:100; Abcam, ab9050), rabbit anti–cleaved Casp3 (1:100; Cell Signaling Technology, 9664S), rabbit anti-TBR2 (1:1000; Abcam, ab23345), and rat anti-BrdU (1:500; Abcam, ab6326)] in blocking buffer overnight at 4°C. After three times of rinsing in PBS, sections were incubated in secondary antibodies (Alexa Fluor 488–conjugated anti-mouse, Alexa Fluor 555–conjugated anti-mouse, Alexa Fluor 488–conjugated anti-rat, Alexa Fluor 488–conjugated anti-rabbit, and Alexa Fluor 555–conjugated anti-rabbit; Thermo Fisher Scientific; 1:1000) for 1 hour at room temperature. Nuclei were labeled by incubation in PBS containing 4′,6-diamidino-2-phenylindole (0.1 μg/ml) (Sigma-Aldrich), and samples were mounted in ProLong Gold Antifade Mountant (Thermo Fisher Scientific).
Alexa fluor 555 conjugated anti rabbit
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 555-conjugated anti-rabbit is a fluorescent-labeled secondary antibody used in various immunodetection techniques. It is designed to bind to primary rabbit antibodies, enabling visualization and detection of target proteins or molecules in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 conjugated anti mouse, by Thermo Fisher Scientific (6 mentions)
Lsm 780, by Zeiss (4 mentions)
P atm ser1981 10h11 e12, by Merck Group (2 mentions)
Anti γh2ax jbw301, by Merck Group (2 mentions)
Lsm 510 meta confocal microscope, by Zeiss (2 mentions)
Ab23345, by Abcam (1 mentions)
Ab9050, by Abcam (1 mentions)
Ab18465, by Abcam (1 mentions)
Ab6326, by Abcam (1 mentions)
Sc 13024, by Merck Group (1 mentions)
Alexa fluor 488 conjugated anti rat, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated anti rabbit, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade mountant, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Fluorescent mounting medium, by Merck Group (1 mentions)
Iba 1, by Abcam (1 mentions)
Tcs sp2, by Leica camera (1 mentions)
Lemosol, by Fujifilm (1 mentions)
Paraformaldehyde (pfa), by Fujifilm (1 mentions)
Ab28364, by Abcam (1 mentions)
9 protocols using alexa fluor 555 conjugated anti rabbit
1
Immunohistochemical Analysis of Cortical Development
2
Immunolabeling and Quantification of Microglia
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The brain sections were blocked for 10 min with 3% BSA in PBS and then incubated for one night at 4 °C by the primary antibodies Iba-1 (Rabbit monoclonal) (1:500; Abcam, USA). Subsequently, the sections were incubated with the secondary antibodies [1:1000 Alexa Fluor 555-conjugated anti-rabbit (Thermo Fisher Scientific, USA)] for 2 h. Three times 10 min rinses in PBS were conducted following each of the aforementioned steps. In the final step, fluorescent mounting medium (Sigma, USA) was applied to the sections. Laser-scanning confocal microscopes (Leica TCS SP2, Germany) were used to capture confocal images. Microglia were analyzed based on their branching lengths and number of branching endpoints as determined in the previous study [28 ].
3
Histological Characterization of Tubular Liver Tissue
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To confirm the quality of the prepared tubular liver tissue, paraffin-embedded tissue sections were prepared for observation. The tissues were fixed with 4% paraformaldehyde (FUJIFILM Wako Pure Chemical) for 6 h at 4 °C, dehydrated with ethanol, permeabilized with Lemosol (FUJIFILM Wako Pure Chemical), and embedded in paraffin. Then, sections of 5 μm thickness were prepared. After deparaffinization by Lemosol and ethanol, the sections were subjected to staining with hematoxylin and eosin (HE) and immunostaining. For immunohistochemistry (IHC), after heat-induced epitope retrieval (HIER) using citrate buffer (pH 6), the sections were incubated with the primary antibody for CD31 (1:100, ab28364, Abcam PLC, Cambridge, UK), followed by a secondary antibody (ImmPRESS Reagent, Anti-Rabbit Ig, Vector Laboratories, Inc., Burlingame, CA, USA). Finally, the sections were colorized with DAB (ImmPACT DAB, Vector Laboratories) and counterstained with hematoxylin. For immunofluorescence, after HIER, the sections were incubated with the primary antibodies against CD31 and CK18 (1:200, MA5-12104, Thermo Fisher Scientific Inc., Waltham, MA, USA) and secondary antibodies (1:200, Alexa Fluor 488-conjugated anti-mouse and Alexa Fluor 555-conjugated anti-rabbit, Thermo Fisher Scientific Inc.).
4
Immunofluorescence Analysis of Endothelial Cell Markers
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The samples were fixed with 4% paraformaldehyde for 10 min at room temperature
after 7 days of culturing. The fixed samples were washed with 1× PBS for 30 min
and then blocked with 0.2% normal goat serum and 0.2% Triton X-100 in PBS for
1 h at room temperature. Immunofluorescence was performed with the following
antibodies: rabbit anti-CD31/PE-CAM (Novusbio, USA, 1:50), rabbit
anti-VE-cadherin (Cell Signaling Technology, USA, 1:50), mouse anti-Zo-1
(ThermoFisher Scientific, USA, 1:50), and mouse anti-claudin 5 (Abcam, England,
1:50). The samples were incubated with the primary antibodies at room
temperature for 1 h and then washed thrice with 1× PBS. Alexa Fluor
488-conjugated goat anti-mouse (ThermoFisher, USA) and Alexa Fluor
555-conjugated anti-rabbit (ThermoFisher, USA) antibodies were used at a
dilution 1:50. 4′, 6-diamidino-2-phenylindole (DAPI) stain was used for nuclear
staining. Immunofluorescence images were obtained using a Nikon ECLIPSE Ti-S
fluorescence microscopy system (Japan).
after 7 days of culturing. The fixed samples were washed with 1× PBS for 30 min
and then blocked with 0.2% normal goat serum and 0.2% Triton X-100 in PBS for
1 h at room temperature. Immunofluorescence was performed with the following
antibodies: rabbit anti-CD31/PE-CAM (Novusbio, USA, 1:50), rabbit
anti-VE-cadherin (Cell Signaling Technology, USA, 1:50), mouse anti-Zo-1
(ThermoFisher Scientific, USA, 1:50), and mouse anti-claudin 5 (Abcam, England,
1:50). The samples were incubated with the primary antibodies at room
temperature for 1 h and then washed thrice with 1× PBS. Alexa Fluor
488-conjugated goat anti-mouse (ThermoFisher, USA) and Alexa Fluor
555-conjugated anti-rabbit (ThermoFisher, USA) antibodies were used at a
dilution 1:50. 4′, 6-diamidino-2-phenylindole (DAPI) stain was used for nuclear
staining. Immunofluorescence images were obtained using a Nikon ECLIPSE Ti-S
fluorescence microscopy system (Japan).
5
Influenza Virus Localization in A549 Cells
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A549 cells grown on coverslips were infected with influenza A WSN virus at a multiplicity of infection (MOI) 2. At the indicated time points, the cells were fixed with 4% paraformaldehyde. After treating the fixed cells with 0.3% Triton X-100, the cells were subjected to immunofluorescence staining, as previously described [24 (link)]. Briefly, the fixed cells were blocked with 5% bovine serum albumin and incubated with monoclonal anti-hnRNP A2/B1 (Sigma-Aldrich) and polyclonal anti-NP antibodies, which were generated in house. The fluorescence-conjugated antibodies (Alexa Fluor 488-conjugated anti-mouse and Alexa Fluor 555-conjugated anti-rabbit; Life Technologies, Carlsbad, CA, USA) were applied to detect the primary antibodies. Nuclei were stained with 4’ 6-diamidino-2-phenylindole (DAPI). The localization of the indicated proteins and nuclei was then observed on a ZEISS LSM510 META confocal microscope.
6
Characterizing Inflammation Modulators in NIH3T3 Cells
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Recombinant human TNF-α, hemin and ATP were purchased from Sigma Aldrich. NIH3T3 cells were treated with reagents diluted in complete medium at concentrations determined by preliminary experiments and detailed below. Anti-hE5NT (4G4, Novus Biologicals), anti-hHO-1 (EP1391Y, Epitomics) and anti-hENTPD1 (BU61, Santa Cruz) primary antibodies, Alexa Fluor 488-conjugated anti-mouse and Alexa Fluor 555-conjugated anti-rabbit (Life Technologies) secondary antibodies were used for immunofluorescence analysis. anti-hHO-1 (EP1391Y, Epitomics), anti-hE5NT (EPR6115, LifeSpan BioSciences), anti-hENTPD1 (HPA014067, Sigma Aldrich), anti-β-actin (AC-15, Sigma Aldrich)), anti-Nf-kB1 p105/p50 (D4P4D, Cell Signaling) and anti-Lamin B2 (EPR9701(B), Abcam) primary antibodies were used for immunoblotting analysis. Phycoerythrin (PE)-conjugated anti-hE5NT (BD Biosciences) and Alexa Fluor 647-conjugated anti-hENTPD1 (Life Technologies) antibodies were used for FACS analysis and cell sorting.
7
TGF-β1 Induces β-arrestin2 and TβRIII Colocalization
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Cells were seeded in a six-well dish with poly-D-lysine-coated coverslips. After incubation overnight, the cells were starved and stimulated with TGF-β1 5 ng/mL (PeproTech, NJ, USA) for the indicated time. The cells were then fixed with 4% paraformaldehyde for 20 min, washed thrice with PBS and permeabilized with 0.1% Triton X-100 for 5 min. After that, the cells were incubated with 1% bovine serum albumin, followed by primary antibodies against β-arrestin2 and TβRIII overnight at 4 °C. The samples were subsequently incubated with a mixture of Alexa Fluor 555-conjugated anti-rabbit and Alexa Fluor 488-conjugated anti-mouse secondary antibodies (Life Technologies Inc., CA, USA) for 2 h in the dark. The samples were then mounted with a sealer containing DAPI, and the images were captured with a Leica SP8 laser scanning confocal microscope (Leica Biosystems, Wetzlar, Germany). β-arrestin2-positive expression is presented as green fluorescent foci, TβRIII-positive expression is presented as red fluorescent foci, and colocalization of these two proteins is presented as yellow fluorescent foci.
8
Immunofluorescent Detection of DNA Damage Markers
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Cells were plated onto sterile glass coverslips and fixed with PBS-PFA 4% (wt/vol) for 15 min at RT and washed twice in PBS. Cells were permeabilized with lysis buffer (sucrose 300 mM, MgCl2 3 mM, Tris, pH 7.0, 20 mM, NaCl 50 mM, and Triton X-100 0.5%) for 3–5 min at RT under slow agitation. The following antibodies were diluted in PBS-BSA 4% and applied to the coverslips for 40 min at 37°C: anti-γH2AX (JBW301, 1:800) and P-ATM Ser1981 (10H11.E12, 1:200) from Millipore. Cells were then incubated with Alexa-Fluor 488-conjugated anti-mouse or Alexa-Fluor 555-conjugated anti-rabbit (1:800; Life Technologies) for 20 min at 37°C and then in Hoechst 33342 (500 ng/ml in PBS) for 10 min at RT. Fluorescence microscopy pictures were taken using a Nikon Eclipse Ni-E microscope, or a confocal Zeiss LSM 780. The Fiji program was used to analyze fluorescence intensity and number of foci per nuclei.
9
Immunofluorescence Assay for DNA Damage
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Cells were plated onto sterile glass coverslips and fixed with PBS-PFA 4% (wt/vol) for 15 min at room temperature (RT) and washed twice in PBS. Cells were permeabilized with lysis buffer (sucrose 300 mM, MgCl 2 3 mM, Tris pH 7.0 20 mM, NaCl 50 mM, Triton X-100 0.5%) for 3-5 min at RT under slow agitation. The following antibodies were diluted in PBS-BSA 4% and applied to the coverslips for 40 min at 37°C: anti-γH2AX (JBW301, 1:800), P-ATM Ser1981 (10H11.E12, 1:200), and 53BP1 (1:500) from Millipore, and anti-MDC1
(1:200) from AbCam. For NLRP3 labeling, cells were fixed with PBS-PFA 4% (wt/vol) for 15 min at RT, washed twice in PBS and permeabilized with 1% triton X100. Anti-flag was diluted in saturation buffer (PBS, 1% BSA; NaCl 0.02%; Tween 20 0.5%; SVF 3%) and incubated on cells for 1 h. Cells were then incubated with Alexa-Fluor 488-conjugated antimouse or Alexa-Fluor 555-conjugated anti-rabbit (1:800; Life Technologies) for 20 min at 37°C and in Hoechst (500 ng/mL in PBS) for 10 min at RT. Fluorescence microscopy pictures were taken using a Nikon Eclipse Ni-E microscope, and confocal Zeiss LSM 780.
(1:200) from AbCam. For NLRP3 labeling, cells were fixed with PBS-PFA 4% (wt/vol) for 15 min at RT, washed twice in PBS and permeabilized with 1% triton X100. Anti-flag was diluted in saturation buffer (PBS, 1% BSA; NaCl 0.02%; Tween 20 0.5%; SVF 3%) and incubated on cells for 1 h. Cells were then incubated with Alexa-Fluor 488-conjugated antimouse or Alexa-Fluor 555-conjugated anti-rabbit (1:800; Life Technologies) for 20 min at 37°C and in Hoechst (500 ng/mL in PBS) for 10 min at RT. Fluorescence microscopy pictures were taken using a Nikon Eclipse Ni-E microscope, and confocal Zeiss LSM 780.
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