As previously described (Ning et al., 2015 (link), 2017 (link)), rabbit anti-SFTSV NSs, NP, and RdRp or HRTV-NSs antiserum were respectively, raised against the corresponding viral proteins generated by Escherichia coli. The antibody anti-β-actin (ABclonal, China) was purchased from the manufacturer. For the secondary antibodies, goat anti-rabbit IgG conjugated with Alexa Fluor 488 (Thermo Fisher Scientific, United States) and goat anti-rabbit IgG conjugated with Alexa Fluor 555 (Thermo Fisher Scientific, United States) used in the IFA assay were purchased from the manufacturer; goat anti-rabbit IgG antibodies conjugated with HRP (Abcam, United States) were used for Western blot and Co-IP analysis.
Goat anti rabbit igg conjugated with alexa fluor 488
Manufactured by Thermo Fisher Scientific
Sourced in United States
Goat anti-rabbit IgG conjugated with Alexa Fluor 488 is a secondary antibody that binds to rabbit primary antibodies. The Alexa Fluor 488 dye is attached to the antibody, allowing for fluorescent detection and visualization.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710, by Zeiss (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Lsm 510 meta, by Zeiss (2 mentions)
Goat anti rabbit igg conjugated with alexa fluor 555, by Thermo Fisher Scientific (1 mentions)
Anti β actin, by ABclonal (1 mentions)
Mouse iggk bp hrp, by Santa Cruz Biotechnology (1 mentions)
4 6 diamidino 2 phenylindole dapi, by Abcam (1 mentions)
Triton x 100, by Bio-Rad (1 mentions)
Rabbit anti alpha smooth muscle actin, by Abcam (1 mentions)
Axio observer microscope, by Zeiss (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Ab79056, by Abcam (1 mentions)
Ab183685, by Abcam (1 mentions)
Goat anti rabbit igg conjugated with alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Sc 12755, by Santa Cruz Biotechnology (1 mentions)
Tissue tek, by Sakura Finetek (1 mentions)
Dm2500 upright microscope, by Leica (1 mentions)
Alexa fluor 555 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Normal goat serum (ngs), by Vector Laboratories (1 mentions)
Goat anti guinea pig igg alexa fluor 555, by Thermo Fisher Scientific (1 mentions)
15 protocols using goat anti rabbit igg conjugated with alexa fluor 488
1
Antibody Production and Characterization for SFTSV
2
Diverse Immunoblotting Antibody Protocols
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The following antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA): Anti-Fra-1 (sc-183) (sc-605x) (sc-28310), anti-c-Fos (sc-52), anti-c-Jun (sc-44) (sc-74543), anti-Jun D (sc-74) (sc-271938), anti-α-Tubulin (sc-8035), normal rabbit IgG (sc-2027), and mouse-IgGk BP-HRP (sc-516102). Goat anti-rabbit IgG HRP (ADI-SAB-300-J) was purchased from Enzo Life Science, Inc. (Farmingdale, NY, USA). ECL Sheep anti-Mouse IgG HRP (NA931V) was from Amersham Bioscience UK limited (Buckinghamshire, UK). IRDye® 800CW Donkey anti-Rabbit IgG (926-32213), IRDye® 680RD Donkey anti-Mouse IgG (926-68072) were from Li-COR Bioscience (Lincoln, NE, USA). Goat anti-Rabbit IgG conjugated with Alexa Fluor 488 was from Thermo Fisher Scientific, Inc. (Invitrogen) (Waltham, MA, USA).
3
Immunocytochemical Characterization of Cells
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Cells of 3–6th passage were attached to L-polylysine-coated glass slides, fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 (Bio-Rad, Hercules, CA, USA) for 20 min at room temperature, and then blocked with 1% bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA) at room temperature for 1 h, and incubated with mouse anti-CD90 (1:100, cat. no. ab134358; Abcam, Cambridge, UK), rabbit anti-alpha smooth muscle actin (1:100, cat. no. A17910; ABclonal, Wuhan, China), mouse anti-fibronectin (1:100, cat. no. ab6328; Abcam, Cambridge, UK) monoclonal antibody conjugated with fluorochrome for 12 h at 4 °C. Then, the cells were incubated for 40 min at room temperature with secondary antibodies (goat anti-mouse IgG conjugated with Alexa Fluor 488, 1:400, cat. no. A11029; Thermo Fisher Scientific, Waltham, MA, USA and goat anti-rabbit IgG conjugated with Alexa Fluor 488, 1:400, cat. no. ab150077; Abcam, Boston, MA, USA) and washed. The cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, Abcam, Cambridge, UK). The actin–cytoskeleton of the cells was stained with phalloidin coupled with Alexa Flour 488 (1:200, cat. no. A12379, Thermo Fisher Scientific, Waltham, MA, USA) for 60 minutes, as per the manufacturer’s instructions. The cells were photographed using an Axio Observer microscope (Carl Zeiss, Oberkochen, Germany).
4
Immunofluorescence Analysis of Ocular CD4+, IL-17+, and IFN-γ+ Cells
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The left eyeballs were embedded in optimal cutting temperature (OCT) compound (Tissue-Tek, catalog no. 4583; Sakura Finetek USA, Torrance, CA, USA). Frozen sections (5 µm thick) were fixed with acetone for 15 minutes at –20°C. Subsequently, the samples were washed three times with PBS, followed by blocking with 2% BSA in PBS for 1 hour at room temperature. The samples were then incubated with monoclonal rabbit anti-CD4 antibody (1:200, 3.475 µg/mL, catalog no. ab183685; Abcam, Inc., Cambridge, MA, USA) and counterstained with polyclonal rabbit anti-IL-17 antibody (1:200, 5 µg/mL, catalog no. ab79056; Abcam) and monoclonal mouse anti-IFN-γ antibody (1:50, 4 µg/mL, catalog no. sc-12755; Santa Cruz Biotechnology, Dallas, TX, USA) diluted with 1% BSA overnight at 4°C. After they were washed three times with PBS, the samples were incubated in secondary antibody (goat-anti-rabbit IgG conjugated with Alexa Fluor 488, goat-anti-rabbit IgG conjugated with Alexa Fluor 594, or goat-anti-rat IgG conjugated with Alexa Fluor 594; Thermo Fisher Scientific) diluted with 1% BSA for 1 hour at room temperature. Finally samples were counterstained with 4′,6-diamidino-2-phenylendole (DAPI, catalog no. H-1200; Vector Laboratories, Inc., Burlingame, CA, USA). The results were photographed with a Leica upright microscope (DM2500; Leica Microsystems, Wetzlar, Germany).
5
Dual Fluorescent Immunolabeling of IL-1β and NLRP Proteins
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Double fluorescent immunolabelings were carried out to determine the co-localization of IL-1β and NLRP proteins with other markers. Before antibody treatments, tissue sections were kept in 10% normal goat serum (Vector Labs) dissolved in PBS for 50 min, then incubated with a selection of several antibodies that contained either (a) rabbit anti-IL-1β, (b) rabbit anti-NLRP1, (c) rabbit anti-NLRP2 (d) rabbit anti-NLRP3 and one of the following antibodies: (e) mouse anti-glial fibrillary acidic protein (GFAP) (diluted 1:500; Chemicon, Temecula, CA, USA; catalog no. MAB3402), (f) guinea-pig anti Iba1 (diluted 1:2000; Synaptic Systems, Goettingen, Germany; catalog No. 234-004). Sections were gently shaken in the primary antibody solutions for 2 days at 4 °C and were further placed into the proper combination of secondary antibodies for 2 h selected from the following: (a) goat anti-rabbit IgG conjugated with Alexa Fluor 488 (diluted 1:1000; Thermo Fisher Scientific, Waltham, MA, USA; catalog No. A11034), (b) goat anti-mouse IgG-Alexa Fluor 555 (diluted 1:1000, Thermo Fisher Scientific, catalog no. A21422), (c) goat anti-guinea-pig IgG-Alexa Fluor 555 (diluted 1:1000, Thermo Fisher Scientific, catalog No. A21435). Sections were covered with mounting medium and Vectashield (Vector Labs) on glass slides.
6
EBV Receptor and Cytoskeleton Expression
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To determine the efficiency of eGFP‐EBV infection, cells were detached using trypsin and suspended in FACS solution (2% FBS in PBS (−)) containing 7‐AAD (eBioscience) to distinguish the dead cells. To determine EBV receptor expression, cells were exposed to H. pylori (MOI 100) for 7 h under cell culture conditions. Cells were fixed with Mildform 10 N (FUJIFILM Wako Pure Chemical Corp.) for 10 min at 25℃. After washing with PBS (−), the cells were incubated with PBS (−) containing 0.5% saponin (Wako) for 20 min at 25℃. Cells were incubated with PBS (−) containing 0.5% saponin and 5% bovine serum albumin fraction V (Sigma‐Aldrich) for 1 h at 25℃. The cells were stained with rabbit anti‐human‐EphA2 antibody (1C1, Novus Biological) or with mouse anti‐human NMHC‐IIA (GT218; Genetex) for 30 min on ice. Cells were then washed with FACS solution and stained with goat anti‐rabbit IgG conjugated with Alexa Fluor 488 or goat anti‐mouse IgG conjugated with Alexa Fluor 488 (Thermo Fisher Scientific) for 30 min on ice. Finally, cells were suspended in FACS solution containing 7‐AAD and analyzed using a FACS Calibur flow cytometer and FlowJo software (FlowJo LLC).
7
Bradyzoite-Induced NETosis and LC3 Expression
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LC3 has been used as a classical marker for autophagosomes (Karim et al., 2007 (link)), being LC3-I cytosolic and LC3-II membrane bound and enriched in the autophagic vacuole. Therefore, we tested whether LC3 expression might be present during B. besnoiti bradyzoite-induced NETosis as described elsewhere (Zhou et al., 2019 (link)). Briefly, bovine PMN (n = 3) were added on 0.01% poly-L-lysine pre-coated coverslips (15 mm diameter; Nunc), then stimulated by B. besnoiti bradyzoites for 1 h at RT. After incubation, cells were fixed with 4% paraformaldehyde for 10 min, permeabilized with cold methanol (Merck) for 3 min and blocked by using the following blocking buffer (5% BSA (Sigma-Aldtich), 0.1% Triton X-100 (Sigma-Aldrich) in sterile PBS) for 60 min at RT. After removing blocking buffer, cells were incubated overnight at 4°C with rabbit anti-LC3B antibodies (Cat#2775, 1:200, Cell Signaling Technology) diluted in blocking buffer, washed three times with PBS, incubated with goat anti-rabbit IgG conjugated with Alexa Fluor 488 (Invitrogen) for 1 h in the dark at RT. After being washed three times with PBS, coverslips were mounted by prolonged anti-fading reagent with DAPI (Invitrogen) on glass slides (Nunc), and five randomly images were taken per condition using an inverted epifluorescence microscope IX 81® (Olympus) and/or by using confocal microscopy analysis (LSM 710®; Zeiss).
8
Visualizing CAR19 and LCK Localization
9
Immunofluorescence Staining of Leishmania infantum
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L. infantum early-logarithmic promastigotes (day 2) were laid on the slides (∼2 × 105 each) and dried at room temperature. The cells were fixed for 5 min at room temperature in 4% paraformaldehyde. Then they were washed in PBS and permeabilized with PBS-0.5% Triton X-100. Blocking was carried out with 5% skimmed milk in PBS-0.1% Tween 20 at 37 °C for 30 min in a wet chamber. The coverslips were incubated with a 1:1000 dilution of the LiTAT polyclonal antibody in blocking buffer for 1 h. After washing, the slides were incubated with a 1:200 dilution of goat anti-rabbit IgG conjugated with Alexa Fluor 488 (Life Technologies, USA) in blocking buffer in darkness for 1 h. During the last 10 min of incubation with the secondary antibody, coverslips were treated with DAPI (Life Technologies) at a final concentration of 0.2 μg/ml in darkness. After washing, the coverslips were mounted on the slides with Mowiol (Merck-Millipore, Germany) and observed in a confocal microscope Leica TCS SP5, with a magnification of 630×.
10
Immunolocalization of AOC in Stem Bases
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Immunocytochemical detection of AOC in stem bases was performed as described [42 (link)]. Small pieces of stems were fixed with 4 % (w/v) paraformaldehyde/0.1 % (v/v) Triton X-100 in phosphate buffered saline (135 mM, NaCl, 3 mM KCl, 1.5 mM KH2PO4, and 8 mM Na2HPO4) and embedded in polyethylene glycol 1500 (Merck, Darmstadt, Germany). Cross-sections of 5 μm thickness were used for immunolabeling with the rabbit polyclonal antibody raised against recombinant LeAOC [40 (link)] at a dilution of 1:1000. The use of pre-immune serum at the same dilutions served as a control and revealed no signals. As secondary antibody, goat anti-rabbit IgG conjugated with AlexaFluor488 (life technologies, Carlsbad, CA, USA) was used according to the manufacturer’s instructions. Sections were analyzed by confocal laser scanning microscopy using a LSM510 META (Carl Zeiss GmbH, Jena, Germany).
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