Tissues were harvested and processed as described above. For p-p38 staining, specimens were treated with rabbit anti-mouse primary antibody (Cell Signaling Technology, USA, 1:800, 4 °C overnight), and goat anti-rabbit IgG (H + L) secondary antibody, Alexa Fluor® 488 conjugate (Thermo Fisher Scientific, USA, 1:200, 37 °C for 1 hour). For neutrophil staining, specimens were treated with rat anti-mouse primary antibody (Anti-Neutrophil Elastase, Abcam Ltd., USA, 1:100, 4 °C overnight), and goat anti-rat IgG (H + L) secondary antibody, Alexa Fluor® 633 conjugate (Thermo Fisher Scientific, USA, 1:200, 37 °C for 1 hour). For KGF-2/FGF-10 staining, sections were treated with sheep anti-mouse primary antibody (R&D Systems, USA, 10 µg/mL, 4 °C overnight), followed by Alexa Fluor 633 conjugated donkey anti-sheep IgG secondary antibody (Thermo Fisher Scientific, USA, 1:200, 37 °C for 1 hour). Images were captured at the wound margin for evaluation (×200 magnification).
Alexa fluor 633 conjugate
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 633 Conjugate is a fluorescent dye used for labeling biomolecules, such as proteins and nucleic acids, in various biological applications. It has an excitation maximum at 632 nm and an emission maximum at 647 nm, making it suitable for detection in the red region of the visible spectrum.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 conjugate, by Thermo Fisher Scientific (2 mentions)
Lysosensor green, by Thermo Fisher Scientific (2 mentions)
Tcs sp5 confocal microscope, by Leica (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Anti neutrophil elastase, by Abcam (1 mentions)
Donkey serum, by Merck Group (1 mentions)
Alexa fluor 594 conjugate, by Thermo Fisher Scientific (1 mentions)
Apoptag plus in situ apoptosis fluorescein detection kit, by Merck Group (1 mentions)
Tppp3, by Abcam (1 mentions)
Mab3324, by R&D Systems (1 mentions)
Bglap, by Abcam (1 mentions)
Ab19355, by Abcam (1 mentions)
Tcs sp8, by Leica (1 mentions)
Lsrfortessa flow cytometer, by BD (1 mentions)
Tcs spe confocal microscope, by Leica (1 mentions)
4 6 diamidino 2 phenylindole dihydrochloride dapi, by Thermo Fisher Scientific (1 mentions)
Mab1420, by R&D Systems (1 mentions)
Ultraview universal alkaline phosphatase red detection kit, by Roche (1 mentions)
Mouse anti β 3 tubulin, by BioLegend (1 mentions)
Rabbit anti egr 1, by Cell Signaling Technology (1 mentions)
9 protocols using alexa fluor 633 conjugate
1
Immunohistochemical Staining of Wound Tissues
2
Lysosomal Trafficking Dynamics with DMFs
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HeLa cells were incubated with 1 μM DMFs at 37 °C with 5% CO2 for different time lengths, followed by washing twice with PBS to remove free probes. LysoSensor Green (Thermo Fisher) was used for lysosome staining by incubating with cells at 37 °C for 30 min and transferrin from Human Serum, Alexa Fluor 633 Conjugate (ThermoFisher) was incubated with cells for 1 hour. Fluorescence imaging was performed on a Leica TCS SP5 confocal microscope (Leica Microsystems) with a 40× oil-immersion objective. In most cases, the optical slice thickness was adjusted to 0.5 μm. DMFs with TAMRA were excited at 543 nm, and the fluorescence was collected at 570 nm. LysoSensor Green was excited at 443 nm, and the fluorescence was collected at 505 nm. Transferrin was excited at 633 nm, and the fluorescence was collected at 650 nm.
3
Lysosomal Trafficking Dynamics with DMFs
4
Immunofluorescence Staining and TUNEL Assay
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Tissue sections or primary cells were fixed and blocked with 5% Donkey serum (Sigma-Aldrich, USA). Samples were then incubated with primary antibodies at 4°C overnight. The following antibodies were used for staining: Bglap (Abcam, USA, ab93876, 5 ug/ml); Tppp3 (Abcam, USA, ab150998, 1:50); Mkx (Lifespan Biosciences Inc., USA, LS-B8063, 1 ug/ml); Gli1 (R&D, USA, MAB3324, 10 ug/ml); Gli2 (R&D, USA, AF3635, 5 ug/ml); pH3 (Santa Cruz, USA, sc-8656-R, 1:200); Donkey anti-rabbit IgG (H + L), Alexa Fluor 594 conjugate (Thermo Fisher Scientific, USA, A21207, 1:500); Donkey anti-rat IgG (H + L), Alexa Fluor 488 conjugate (Thermo Fisher Scientific, USA, A21208, 1:500); Donkey anti-goat IgG (H + L), Alexa Fluor 488 conjugate (Thermo Fisher Scientific, USA, A11055, 1:500); and Goat anti-rabbit IgG (H + L), Alexa Fluor 633 conjugate (Thermo Fisher Scientific, USA, A21072, 1:500). Samples were washed with PBS and incubated with secondary antibodies at room temperature for 1 hr. Nuclei were stained with Hoechst 33342 (Thermo Fisher Scientific, USA). Signals were detected under fluorescence microscopy. For TUNEL assay, procedures were followed as described by the manufacturer using the ApopTag Plus In Situ Apoptosis Fluorescein Detection Kit (Millipore, USA, s7111).
5
Immunofluorescence Analysis of Tas2r Expression
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HEK293 (PEAKrapid) cells were seeded onto poly-lysine coated coverslips in 12-well plates and transfected with a Tas2r construct (1 µg/well for each construct), along with Gα16-gust44 (1 µg/well) by Lipofectamine (4 µl/well). After 24 h of transfection, cells were washed twice with warm phosphate-buffered saline (PBS) and placed at 4 °C for 2 h. The cell surface staining was performed by incubation with concanavalin A, Alexa Fluor 633 Conjugate (C21402, Thermo Fisher, 1:5) for 1 h, followed by three rinses with ice cold PBS buffer. The cells were then fixed by 4% paraformaldehyde in PBS for 20 min. Cells were washed three times with PBS and incubated with 0.1% Triton X-100 in PBS for 10 min. After washed with PBS, cells were incubated for 1 h in 10% fetal bovine serum in PBS to block unspecific binding. Next, an anti-HSV antibody (ab19355, Abcam, 1:500) in PBS supplemented with 10% fetal bovine serum was applied overnight at 4 °C. A donkey anti-rabbit secondary antibody conjugated with Alexa Fluor 488 (ANT024, Wuhan antgene Biotechology, 1:1,000) was then used for detection of the HSV tag. The nucleus was stained with 4′,6-diamidino-2-phenylindole (DAPI). Images were captured with confocal laser scanning microscopy (Leica TCS SP8). To evaluate the expression level of Tas2r in HEK293 cells, three independent areas were counted.
6
Transferrin-Uptake Assay in HCC Cells
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The transferrin-uptake assay was performed as previously described73 (link). HCC cells were exposed to 21 and 1% O2 for 48 h. Cells were serum-starved for 30 min and were harvested by trypsinization. Cells were resuspended in serum-free media and incubated with 50 µg/mL ice-cold transferrin, Alexa Fluor™ 633 Conjugate (T23362, Invitrogen) on ice for 10 min and at 37 °C for 10 min. Cells were washed with ice-cold serum-free media, PBS, and acidic buffer (0.1 M glycine, 150 mM NaCl, pH 3.0) and resuspended in ice-cold PBS for flow-cytometry analysis using BD LSRFortessaTM flow cytometer (BD Biosciences). Data were analyzed by FlowJo v10.7 software (FlowJo, LLC).
7
Immunocytochemistry Assay for SGLT1/2
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ECs were cultured on 8-well Lab-Tek® chambers and exposed to either H2O2 (100 µM) or Ang II (100 nM) for 24 h. Cells were fixed during 30 min with 4 % (w/v) paraformaldehyde and then incubated with blocking/permeabilizing buffer (PBS containing 1 % BSA (w/v) and 0.5 % Triton X-100 (w/v)) for 30 min at room temperature. After buffer removal, cells were incubated with 1:100 dilution of either rabbit anti-SGLT1 or SGLT2 for 1 h at 4 °C. After washing 3 times with PBS, they were further incubated with a 1:250 dilution of a polyclonal goat anti-rabbit immunoglobulin G coupled to CF 633 (Alexa Fluor 633 conjugate, Invitrogen) for 1 h at room temperature in the dark. After washing 3 times with PBS, cells were incubated with 1 mg/ml 4’,6-diamidino-2’-phenylindole dihydrochloride (DAPI, Thermo Fisher) during 3 min at room temperature, in order to counterstain nuclei. After disassembling, slides were mounted with fluorescent mounting medium. Images were acquired using a Leica TCS SPE confocal microscope.
8
Immunohistochemical Analysis of Piezo2 in Cardiac Tissue
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Rat EHTs, rat hearts or mouse hearts were fixed for at least 24 h in a phosphate-buffered solution containing 4% formaldehyde stabilized with methanol. After embedding in paraffin, 4–5 µm sections were cut (for EHTs longitudinally in the median plane). Staining conditions for piezo2 were optimized on rat heart tissue samples and worked for mouse and rat tissues. After antigen retrieval (30 min [IHC] or 15 min [IF] in citrate buffer, pH 6.0), rabbit anti-piezo2 polyclonal antibody (Sigma Life Science, HPA031974) at a dilution of 1:1000 [IHC] or 1:100 [IF], mouse anti-troponin I monoclonal antibody (Merck, MAB 1692) at 1:1000 [IF], mouse anti-α-smooth muscle actin monoclonal antibody (R&D, MAB1420) at 1:200 [IF] or IgG isotype (as a control) was employed. Nuclei were stained with hematoxylin [IHC] or DAPI 1:1000 [IF] and cell membranes with wheat germ agglutinin, Alexa Fluor 633 Conjugate (Invitrogen, W21404) 1:400 [IF].
Antibodies were either visualized with the multimer technology-based UltraView Universal Alkaline Phosphatase Red Detection Kit (Roche, 760–501 for IHC) or with goat anti-rabbit Alexa Fluor 488 (Invitrogen, 11034)/goat anti-mouse Alexa Fluor 546 (Invitrogen, 11003) for IF at a dilution of 1:100.
Antibodies were either visualized with the multimer technology-based UltraView Universal Alkaline Phosphatase Red Detection Kit (Roche, 760–501 for IHC) or with goat anti-rabbit Alexa Fluor 488 (Invitrogen, 11034)/goat anti-mouse Alexa Fluor 546 (Invitrogen, 11003) for IF at a dilution of 1:100.
9
Multimodal Imaging of Cellular Markers
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Samples were fixed by incubating with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 15 min at room temperature. After thorough washing with PBS, the fixed cells were permeabilized and blocked with Triton X-100 (0.3 %) and bovine serum albumin (3%) in PBS for 35 min at room temperature (RT). Then, the samples were incubated at 4°C for 48 hours with the following primary antibodies: mouse anti-tubulin β3 (1:1000; BioLegend), rabbit anti-GFAP (1:1000; Abcam), rabbit anti-Egr1 (1:1000; Cell Signaling Technology), and rabbit anti–β-catenin (Cell Signaling Technology). After washing with PBS, the resulting samples were stained with goat anti-rabbit immunoglobulin (IgG) [heavy and light chains (H + L)] secondary antibody or Alexa Fluor 546 (Invitrogen), goat anti-mouse IgG (H + L) secondary antibody, and Alexa Fluor 633 conjugate (Invitrogen) for 40 min at RT. Nuclei were labeled with 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich), and F-actin was labeled with Alexa Fluor 546 phalloidin (Thermo Fisher Scientific) if necessary. All fluorescence images were taken using Prairie Technologies 2-photon and confocal microsope, QuantEM 512SC camera, 60× and 40× objective lenses, and native Prairie View software and visualized by z-stack mode. All the image analysis and processing were performed with ImageJ and Imaris.
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