Anti-FLAG antibody-coated plate (GenScript, L00455C), recombinant His-tagged TS5-p45, and FLAG-tagged NCL-FL and Ctrl overexpression WCL were used in this experiment. Specific FLAG-tagged NCL-FL capture was first verified by titrating the Anti-FLAG antibody-coated plate with increasing doses of NCL-FL lysate. Anti-NCL primary antibody (Abcam, ab22758) and Alexa Fluor 488 conjugated anti-rabbit IgG (Invitrogen, A-11034) were used for detection. The same protocol was applied to Ctrl lysates as a negative control. Saturation binding experiment was then performed by titrating TS5-p45 over FLAG-tagged NCL-FL- and Ctrl-captured wells at 4 °C for overnight incubation. The following day, treated wells were incubated with anti-His (Abcam, ab9108) at room temperature for 2 h and Alexa Fluor 488 conjugated anti-rabbit IgG (Invitrogen, A-11034) at room temperature for 1 h. Finally, fluorescence intensity was measured with Hidex Sense Microplate Reader (Hidex, Finland). Binding affinity was calculated with GraphPad Prism 8.
Alexa fluor 488 conjugated anti rabbit igg
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
Alexa Fluor 488-conjugated anti-rabbit IgG is a secondary antibody used for detection and visualization in various immunological techniques. It is a conjugate of an anti-rabbit IgG antibody and the Alexa Fluor 488 fluorescent dye. Alexa Fluor 488 is a green-fluorescent dye with excitation/emission maxima of 495/519 nm.
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Lab products found in correlation
Alexa fluor 488 conjugated anti mouse igg, by Thermo Fisher Scientific (13 mentions)
Alexa fluor 594 conjugated anti mouse igg, by Thermo Fisher Scientific (12 mentions)
Alexa fluor 555 conjugated anti mouse igg, by Thermo Fisher Scientific (12 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (10 mentions)
Alexa fluor 568 conjugated anti mouse igg, by Thermo Fisher Scientific (10 mentions)
Lsm 700, by Zeiss (8 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (7 mentions)
Triton x 100, by Merck Group (7 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (6 mentions)
Alexa fluor 594 conjugated anti rabbit igg, by Thermo Fisher Scientific (5 mentions)
Alexa fluor 555 conjugated anti rabbit igg, by Thermo Fisher Scientific (4 mentions)
Cy3 conjugated anti mouse igg, by Jackson ImmunoResearch (4 mentions)
A21206, by Thermo Fisher Scientific (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (4 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (4 mentions)
Hoechst 33342, by Thermo Fisher Scientific (4 mentions)
Ab19027, by Abcam (3 mentions)
Vectashield hardset mounting medium with dapi, by Vector Laboratories (3 mentions)
Vectashield mounting medium, by Vector Laboratories (3 mentions)
Lsm 510 meta, by Zeiss (3 mentions)
172 protocols using alexa fluor 488 conjugated anti rabbit igg
1
Quantifying NCL-TS5-p45 Binding Affinity
2
Immunocytochemistry of Microglia and Markers
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BV2 cells and primary microglial cells were seeded into uncoated glass coverslips in 24-well culture plates. PFA-fixed cells were washed with PBS and incubated with permeabilization buffer (0.1 M PBS and 0.2% Triton X-100) for 15 min at RT. Then, cells were incubated with blocking buffer (0.1 M PBS, 0.2% Triton X-100, and 1% BSA) for 1 h at RT and treated with Iba1 (1:500, 019-19741; Wako), DSCR1 (1:100, RP3941; ECM Biosciences), and CD68 (1:200, ab53444; Abcam) primary antibodies in blocking buffer at 4°C overnight. The primary antibodies were washed with PBS, and then, the cells were immersed in Alexa Fluor 488–conjugated anti-rabbit IgG (1:500; Invitrogen) or Alexa Fluor 647–conjugated anti-rat IgG (1:500; Invitrogen) secondary antibodies for 2 h at RT. After washing the secondary antibodies with PBS, coverslips containing seeded cells were mounted on glass slides with mounting solution (Invitrogen).
3
Immunohistochemical Analysis of Brain Sections
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After behavior test, animals were intracardially perfused with PBS followed by 4% PFA as previously described37 (link),38 (link). Briefly, the brain sections were cut and incubated at room temperature in PBS with 0.01% Triton X-100 for 30 min and followed by blocking with 3% bovine serum albumin for 1 h. For immunolabeling, brain slices were probed with primary antibody overnight at 4 °C. Antibodies included phospho-CaMKII (Thr286) (1:200)39 (link),40 (link), laminin, GFAP, OX-42, and S100β (1:500, Millipore, U.S.A), Nuclei were stained with DAPI (4, 6-diamidino-2- phenylindole) (Sigma-Aldrich, U.S.A). After washing, the sections were incubated with Alexa Fluor 488-conjugated anti-rabbit IgG and Alexa Fluor 594-conjugated anti-mouse IgG (Invitrogen, Carlsbad, CA). Signals were visualized by using a Zeiss LSM 510 confocal microscope. The relative fluorescence intensity of immunostaining was quantified by using Image J software (NIH, Bethesda, MD, USA).
4
Evaluating GRP94 IgG Binding to Colorectal Cancer Cells
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To evaluate GRP94 IgG binding to CRC cells, 2 × 105 HCT116, HT29, Lovo, HCT-8, and Caco-2 CRC cells were fixed with 4% (w/v) paraformaldehyde (PFA), blocked with PBS containing 1% (w/v) BSA, and stained with 20 µg/mL GRP94 IgG for 1 h. Cells were incubated with Alexa Fluor 488-labeled anti-human Fab antibody (1:1000; Invitrogen) for 1 h. The effects of GRP94 IgG on endothelial cell activation were evaluated by incubating 2 × 105 HUVECs in the presence or absence of 20 ng/mL human tumor necrosis factor-α (Millipore) or 20 μg/mL GRP94 IgG for 24 h. After blocking with PBS containing 1% (w/v) BSA for 1 h at room temperature, cells were incubated with 2 μg per well of anti-intercellular cell adhesion molecule-1 (ICAM-1) or anti-vascular cell adhesion molecule-1 (VCAM-1) antibody for 1 h at 37 °C and then with Alexa Fluor 488-conjugated anti-rabbit IgG (1:1000; Invitrogen) for 1 h at 37 °C. Samples were analyzed by flow cytometry (FACSCalibur, BD Bioscience). Data were analyzed using FlowJo software (TreeStar, Ashland, OR, USA).
5
Immunofluorescence Microscopy of AHSV
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BSR cells were seeded on 12 mm diameter round coverslips in 24-well plates and infected with AHSV at a MOI of 2 or 3. Cells were fixed with 4% paraformaldehyde (PFA) and permeabilised with 0.2% Triton X-100 (Merck Millipore) in PBS. After one hour at room temperature in blocking solution (5% milk powder in PBS) coverslips were incubated overnight in primary antiserum diluted 1:100 (anti-NS4) or 1:250 (anti-fibrillarin) in 1% blocking solution. For secondary labeling, cells were incubated with Alexa Fluor-488 conjugated anti-rabbit IgG or Alexa Fluor-633 conjugated anti-mouse IgG (Invitrogen) at a 1:250 dilution. Nuclei were stained with for ten minutes using 5 mg/mL DAPI (4’,6-diamidino-2-phenylindole, Life Technologies) diluted 1:1000 in 1% blocking solution. Coverslips were mounted on microscope slides in VectaShield mounting medium (Vector Laboratories) and the edges sealed. Samples were viewed with a Zeiss LSM510 Meta Laser Scanning Confocal Microscope.
6
Immunostaining of Cell Cycle Proteins
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Cells were grown on glass coverslips, fixed in methanol:acetone (1:1) for 10 min. After blocking using 3% BSA, the coverslips were incubated with primary antibodies. Primary antibody dilutions used were 1:100 for Aurora-B, and 1:200 for H3 p-S10 and Cyclin A. Alexa Fluor 488-conjugated anti-rabbit IgG and Alexa Fluor 555-conjugated anti-mouse IgG, or Alexa Fluor 488-conjugated anti-mouse IgG and Alexa Fluor 555-conjugated anti-rabbit IgG were used as secondary antibodies (Invitrogen). DNA was visualized using 4′,6-diamidino-2-phenylindole (DAPI) staining. Immunostaining of the cell preparations was recorded using an epifluorescence Zeiss Axioplan 2 microscope (Zeiss, Inc., Thornwood, NY), with Plan-Neofluar 40×/0.75 Ph2 and 63x Plan-Apochromat 63×/1.4 NA oil-immersion objective lenses, attached to a charge-coupled-device camera.
7
In Situ Immunofluorescence Analysis of Caspase-3 in hPSCs
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hPSCs were analyzed for in situ immunofluorescence as previously described51 (link),53 (link). Briefly, cells were rinsed with ice-cold PBS and fixed in PBSA (PBS with 0.1% bovine serum albumin) with 4% formaldehyde for 45 min. After two washes cells were permeabilized with 0.1% Triton X-100 in PBSA with 10% normal goat serum for 30 min, washed twice and stained with a rabbit polyclonal antibody anti-active CASPASE-3 (ab13847, ABCAM). Fluorescent secondary antibody Alexa Fluor 488-conjugated anti-rabbit IgG was purchased from Invitrogen and was used to localize the antigen/primary antibody complexes. Cells were counterstained with DAPI and examined under a NIKON ECLIPSE TE2000-S inverted microscope equipped with a 20X E-Plan objective and a super high-pressure mercury lamp. The images were acquired with a NIKON DXN1200F digital camera, which was controlled by the ECLIPSENET SOFTWARE (version 1.20.0 build 61).
8
Immunofluorescence Characterization of Cardiac Cells
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Immunofluorescence was performed with either EBs or ESC-CMs. Cells were fixed with methanol at -20 ºC for 15min, followed by permeabilization with 0.05% Triton-100. The cells were blocked with 10% FBS for 30 min at room temperature and incubated in PBS containing one of the primary antibodies overnight at 4ºC and followed by secondary antibodies for 2 hours at room temperature. The primary antibodies included mouse monoclonal anti-α-Actinin (Sigma Aldrich, A-7811, USA), rabbit polyclonal anti-Nkx2.5 (Santa Cruz, sc-376565, USA), rabbit polyclonal anti-desmoplakin I/II (Santa Cruz, sc-33555, USA), rabbit polyclonal anti-N-cadherin (Santa Cruz, sc-31030, USA) or rabbit polyclonal anti-Connexin 43 (Abcam, ab-11370, USA), mouse monoclonal anti-Connexin 43 (Abcam, ab-79010, USA), rabbit polyclonal anti-MLC-2v (Abcam, ab-79935, USA). The second antibodies included Alexa fluor 488-conjugated anti-mouse IgG (Invitrogen, Carlsbad, CA, USA), Alexa fluor 594-conjugated anti-mouse IgG (Invitrogen, USA), Alexa fluor 488-conjugated anti-rabbit IgG (Invitrogen, Carlsbad, CA, USA) or Alexa fluor 594-conjugated anti-rabbit IgG (Invitrogen, USA). After washing, the fluorescence images were taken by fluorescence inverted microscope or an Olympus IX81-FV1000 inverted multiphoton laser confocal microscope 24 (link).
9
Immunohistochemical Analysis of Cecum Tissue Sections
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Cecum tissue sections (5 µm) were deparaffinized and rehydrated using xylene and ethanol, respectively. Following antigen retrieval in citrate buffer (10 mM, pH = 6), samples were blocked in normal goat serum (5%) or donkey serum (5%). For immunohistochemistry, sections were stained with mucin 2 (Santa Cruz), claudin 3 (Abcam), p-MLKL (Abcam), Ly-6G/Ly-6c (BioLegend), and F4/80 (BioLegend) antibodies. Subsequently, specific staining was detected using the UltraSensitive S-P Kit and DAB Detection Kit (Maixin-Bio, China) according to the manufacturer’s directions. For immunofluorescence, tissue sections were stained with rabbit-anti-PCNA (Santa Cruz) and Alexa Fluor® 488-conjugated anti-rabbit IgG (Invitrogen). Epithelial cell apoptosis was analyzed by TUNEL staining using a commercial kit (KeyGEN Biotech). DAPI (1 µg/ml) was used to stain nuclei.
10
Zika Virus Infection in SUN2 Mutant Cells
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Control, SUN2KO, SUN2RES, SUN2ΔN+TM, SUN2ΔCC, and SUN2ΔSUN cells were infected with ZIKV at an MOI of 8, and fixed at 24 h p.i.. Control and SUN2KO cells were infected with SFV at an MOI of 3 and fixed at indicated times. Briefly, cells were fixed with 4% PFA after 3 washes with PBS, and permeabilized with 0.02% Triton-X 100 for 15 minutes. Then, cells were blocked with 5% BSA for 30 minutes, followed by incubation with primary antibody at 4 °C overnight. After washes, cells were incubated with Alexa Fluor 647-conjugated goat anti-mouse-IgG (Invitrogen) or Alexa Fluor 488-conjugated anti-rabbit-IgG (Invitrogen) in PBS at room temperature (RT) for 1 h. Cells were then stained with TRITC Phalloidin (Yeasen) diluted in PBS for 45 minutes, followed by incubation with DAPI (Invitrogen) diluted in PBS for 20 minutes. Fluorescence images were acquired with a Nikon C2 microscope and analyzed with the NIS Elements software.
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