For cryosectioning, excised tissue was embedded and flash-frozen in O.C.T medium (Tissue Tech) using a dry-ice slurry/2-methylbutanol mixture and cryosectioned between 8-10um. Tissue cryosections were washed with PBS and fixed with 4% PFA for 10 min. Post fixation, slides were washed three times for 5 min each with PBS followed by permeabilization using 0.2% Triton-X100 in PBS (PBT) for 10 min. Slides were then blocked (5% BSA, 2% normal goat serum in PBT) at room temperature for 1hr. Post block, tissue sections were then incubated in primary antibody overnight at 4°C in blocking solution (3% BSA, 0.2% Triton-X100 in PBS). Primary antibodies used include pan-Laminin (Abcam, #ab11575), Acta2 (Sigma, #A2547), F4/80 (Invitrogen, #MF48000), Cdh1 (BD Bioscience, #610181), and ZO-1/Tjp1 (Abcam, #ab221547). After overnight incubation, slides were washed with PBS, and then incubated with goat ALEXA-Fluor 594- or ALEXA-Fluor 488- conjugated antibodies (Thermo Fisher Scientific) for 2 h at room temperature in blocking solution. Cell nuclei were counterstained with DAPI (Molecular Probes) and mounted in 4% (w/v) propyl gallate anti-fade solution. Immunofluorescent images were obtained using an SP5 confocal microscope (Leica). Acta2 and Cdh1 confocal image quantification analysis was performed as noted above (Okamura et al., 2014 (link); Pennathur et al., 2015 (link)).
Alexa fluor 488 conjugated antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States, France
Alexa Fluor 488-conjugated antibody is a fluorescently labeled antibody used for various research applications. It consists of an antibody covalently linked to the Alexa Fluor 488 dye, a fluorescent molecule that emits green light when excited by a suitable light source. This product can be used for techniques such as flow cytometry, immunofluorescence, and western blotting to detect and visualize target proteins or cellular components.
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Lab products found in correlation
Lsm 710 confocal microscope, by Zeiss (12 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (10 mentions)
Hoechst 33342, by Thermo Fisher Scientific (9 mentions)
Rhodamine phalloidin, by Thermo Fisher Scientific (7 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (5 mentions)
Lsm 700, by Zeiss (4 mentions)
Ab32084, by Abcam (4 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (4 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (3 mentions)
Alexa fluor 594 conjugated antibody, by Thermo Fisher Scientific (3 mentions)
Lsm 780 confocal microscope, by Zeiss (3 mentions)
Vectashield mounting medium, by Vector Laboratories (3 mentions)
Lsm 510 meta, by Zeiss (3 mentions)
710 confocal microscope, by Zeiss (3 mentions)
Vectastain elite kit, by Vector Laboratories (2 mentions)
Alexa fluor 555 conjugated antibody, by Thermo Fisher Scientific (2 mentions)
Alexafluor 568 conjugated antibodies, by Thermo Fisher Scientific (2 mentions)
Alexafluor 680 conjugated antibodies, by Thermo Fisher Scientific (2 mentions)
Evos m5000, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (2 mentions)
98 protocols using alexa fluor 488 conjugated antibody
1
Cryosectioning and Immunofluorescence Analysis
2
Histological Analysis of Lung and Kidney Tissues
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Removed lung and kidney tissues were fixed in 10% buffered formalin overnight, and embedded in paraffin. Lung tissues were cut into 5 µm sections, and stained with hematoxylin and eosin (H&E). Paraffin-embedded sections were de-paraffinized in xylene, dehydrated in ethanol and rehydrated in distilled water. To determine GN, mouse renal tissues were analyzed by Periodic acid-Schiff (PAS) staining [26 (link)]. For TUNEL staining, de-paraffinized lung sections were treated by proteinase K to reactivate antigens, re-fixed by 4% formaldehyde, incubated with equilibrate buffer, and finally labelled by TUNEL detection cocktail [5 (link), 26 (link)]. TUNEL-positive cells were determined by averaging the number from 3 fields (× 400) of positively stained cells with the highest density in each section. Cell nuclei were counterstained with DAPI. Fluorescence was detected by confocal microscopy. For detecting the expression of CitH3, de-paraffinized human or mouse lung sections were stained with anti-CitH3 antibodies, followed by Alexa Fluor 488-conjugated antibodies (Thermo Fisher Scientific). Cell nuclei were counterstained with Hoechst 33258. Fluorescence was detected by confocal microscopy.
3
Triple Immunofluorescence Staining for Cytoskeleton, Inflammation, and Signaling
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Triple IF staining for Cytokeratins (CKs)/TLR4/pSTAT3 was performed on cell cytospins. Briefly, cells were fixed with PBS/Formaldehyde 3.7% and permeabilized with PBS/Triton X-100 0.1%. A phospo-STAT3 antibody (Y705) (1:25) (R&D Systems, Minneapolis, MN, USA) was incubated for 1 h, at room temperature (RT), followed by the corresponding secondary antibody, Alexa fluor 555 Anti-Rabbit (1:300), incubated for 45 min at RT. An Alexa Fluor 647-conjugated ΤLR4 antibody (1:50) (Clone: mouse 76B357.1, Novus Biologicals, LLC, Centennial, CO, USA) was incubated overnight at 4 °C. Two different Alexa Fluor 488-conjugated antibodies for CKs (Clones: mouse AE1/AE3 (1:100), Thermo Fisher Scientific, Waltham, MA, USA) and mouse C11 (1:200) (Novus Biologicals) were also included in the overnight incubation, as previously described. Cell nuclei were detected using DAPI antifade (Invitrogen).
4
Flow Cytometric Analysis of EphA3
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Cell surface expression of EphA3 was analysed using the IIIA4 antibody (in-house, 1:100). IgG1 isotype control antibody (Abcam, 1:100) was used to indicate background staining. Cells were washed twice in ice-cold PBS and blocked for 20 min in 1% bovine serum albumin (BSA) in PBS on ice. After blocking, cells were incubated with primary antibodies for 20 min on ice, washed and incubated with secondary Alexa Fluor 488-conjugated antibodies for 20 min on ice in the dark (ThermoFisher Scientific). EphA3 expression levels were examined using a LSR Fortessa flow cytometer (BD, Franklin Lakes, NJ, USA) and acquired data analysed using FlowJo (FlowJo, LLC, CA, USA) software.
5
Histological Analysis of Lung and Kidney Tissues
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Removed lung tissues were fixed in 10% buffered formalin overnight, and embedded in paraffin. Lung tissues were cut into 5 µm sections, and stained with hematoxylin and eosin (H&E). Paraffin-embedded sections were de-paraffinized in xylene, dehydrated in ethanol and rehydrated in distilled water. To determine glomerulonephritis (GN), mouse renal tissues were analyzed by Periodic acid-Schiff staining [35 (link)]. For TUNEL staining, de-paraffinized mouse or human lung sections were treated by proteinase K to reactivate antigens, re-fixed by 4% formaldehyde, incubated with equilibrate buffer, and finally labelled by TUNEL detection cocktail [15 (link)]. Cell nuclei were counterstained with DAPI. For detecting the expression of CitH3 or LC3, de-paraffinized mouse or human lung sections were stained with anti-CitH3 or anti-LC3 antibodies, followed by Alexa Fluor 488-conjugated antibodies (Thermo Fisher Scientific), while cell nuclei were counterstained with Hoechst 33258 [24 (link)].
Their fluorescence was detected by a confocal microscope. Cells with positive TUNEL, colocalized CitH3 with DNAs or cytoplasmic LC3 were determined by averaging the number from 3 fields of positively stained cells with the highest density in each section.
Their fluorescence was detected by a confocal microscope. Cells with positive TUNEL, colocalized CitH3 with DNAs or cytoplasmic LC3 were determined by averaging the number from 3 fields of positively stained cells with the highest density in each section.
6
Characterization of Undifferentiated Adipose-Derived Stem Cells
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Immunostaining was performed on undifferentiated stem cells (uADSCs) at passage 2 cultured on LabTek™ (Nunc) slides. After blocking with normal serum, the primary antibodies were applied for 2 h at room temperature CD73 (1:100; BD Biosciences), CD90 (1:1000; Millipore), CD105 (1:20; Abcam Ltd) and CD34 (1:50; Santa Cruz Biotechnology). After rinsing in phosphate-buffered saline, either secondary goat anti-mouse or donkey anti-goat Alexa Fluor 488 conjugated antibodies (1:300; ThermoFisher) were applied for 1 h at room temperature in the dark. The slides were then cover-slipped with ProLong mounting media containing 4–6-diamido-2-phenylindole (DAPI; ThermoFisher). The specificity of staining was tested by omission of the primary antibodies. To confirm multi-potency the uADSCs were treated with either adipogenic or osteogenic supplements according to the protocol described by the manufacturer (Rat Mesenchymal Stem Cell Functional Identification Kit, R & D Systems). Stem cells which were induced to a Schwann cell-like phenotype were immunostained with Sox-10 (1:200; R&D Systems), S100 protein (1:2000; Dako) and glial fibrillary acidic protein (GFAP 1:1000; Dako) antibodies. For comparison, uADSCs and primary Schwann cells were stained under identical conditions.
7
Biomarker Immunoassays for Research
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IL-2 and TNF-α enzyme-linked immunosorbent assay (ELISA) kits were purchased from Koma Biotech (Seoul, Korea). IgM and IgA ELISA kits were purchased from Affymetrix/eBioscience (San Diego, CA, USA). A cortisol ELISA kit was purchased from Enzo Life Sciences (Farmingdale, NY, USA). Antibodies against CD4, CD8, IL-6, and glyceraldehyde 3-phosphate dehydrogenase were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-p-NF-κB and anti-p-p38 antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Alexa Fluor 488-conjugated antibodies and Hoechst 33342 were obtained from Thermo Fisher Scientific (San Jose, CA, USA). Unless noted otherwise, all chemicals were purchased from Sigma Chemical Co. (St Louis, MO, USA).
8
Immunofluorescence Analysis of T2R14 Expression
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BxPC-3, MiaPaCa-2, PANC-1, SU.86.86, T3M4, MCF-7 and HPDE cell lines were seeded on coverslips in 24-well culture plates and examined at 80-90% confluence. First, cells were washed with PBS and fixed for 10 min at room temperature using 4% paraformaldehyde (Morphisto GmbH). For permeabilization, the cells were treated with 1% bovine serum albumin (BSA)/PBS-Triton X-100 (all from MilliporeSigma). After blocking with 5% BSA for 1 h at room temperature, the cells were incubated overnight at 4°C with T2R14 primary antibody or rabbit IgG isotype control (both 1:1,000). The following day, cells were washed repeatedly with PBS and were then incubated with secondary fluorochrome-conjugated antibodies for 2 h at room temperature. For wild-type cells, Alexa Fluor 488-conjugated antibodies (1:1,000) and Texas Red™-X Phalloidin (1:500; Thermo Fisher Scientific, Inc.) were used. For knockdown cell lines, Alexa Fluor 568-conjugated anti-bodies (1:1,000) were used. The cells were finally washed in PBS repeatedly. The nuclei were stained using fluoroshield DAPI mounting medium (MilliporeSigma). Image acquisition was performed on a Leica TCS SP5 confocal microscope (Leica Microsystems GmbH).
9
Histological and Immunohistochemical Analysis of Liver Tissue
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Liver tissue specimens were embedded in paraffin, and 4‐μm‐thick sections were prepared and stained with H&E. To detect collagen fibers, sections were stained with Sirius red using a Picrosirius Red Stain Kit (Polysciences). For immunohistochemistry, antibodies against Luciferase (Novus Biologicals, NB100‐1677), Ki67 (SP6; Abcam, ab16667), COL1A1 (E8F4L; Cell Signaling, #72026), TAGLN (Abcam, ab14106), F4/80 (Cl:A3‐1; Serotec, MCA497), and phosphorylated Stat3 (Tyr705; D3A7; Cell Signaling, #9145), were used as the primary antibodies. Staining signals were visualized using a Vectastain Elite Kit (Vector Laboratories). For fluorescent immunohistochemistry, antibodies against GFP (1E4; MBL, M048‐3) and RFP (Rockland Immunochemicals, 600‐401‐379) were used to detect Venus‐labeled and tdTomato‐labeled organoid cells, respectively. Alexa Fluor 594‐ or Alexa Fluor 488‐conjugated antibodies (Thermo Scientific, A21202 or A21207) were used as the secondary antibodies.
The mean Ki67‐labeling indices were calculated by counting the positive cells in 15–20 independent lesions (n = 3 mice for each genotype).
The mean Ki67‐labeling indices were calculated by counting the positive cells in 15–20 independent lesions (n = 3 mice for each genotype).
10
Lentiviral Knockdown of LETN in NPCs
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Two days after plating, the induced NPCs were affected with lentivirus carrying mock sequence and coding sequence of shRNA against LETN. Four days after infection, the NPC cultures were fixed with 4% polyformaldehyde. Then the cells were blocked with blocking buffer (5% BSA, Triton X-100 in PBS), and incubated with anti-Ki67 antibody (Thermo #PA5-19462) 1:500 and Alexa Fluor 488-conjugated antibodies (Thermo #R37116). The nucleus was counterstained with DAPI.
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