Cells were fixed with 4% paraformaldehyde for 20 mins, permeabilized and blocked with PBS with 0.3% Triton-X100 and 1% bovine serum albumin for 40 mins, then incubated with primary antibodies diluted with blocking solution at 4 °C overnight. Alexa Fluo series of second antibodies (Thermo Scientific) were applied accordingly for one hour at room temperature. Cells were finally mounted in 4′,6-diamidino-2-phenylindole (DAPI) and examined using fluorescence microscope (Leica DMi8). For BrdU-labeled cells, cells were fixed with cold methanol for 15 mins on ice, rehydrated with PBS for 10 mins, incubated with 2 M hydrochloric acid for 45 mins and neutralized with sodium borate for 15 mins before first antibody incubation. The first antibodies include rabbit anti-active caspase 3 (a-cas3), rabbit anti-BNP, rabbit anti-ANP, mouse anti-sarcomeric α-actinin (SAA; all from Abcam), rabbit anti- discoidin domain-containing receptor 2 (DDR2; Immunoway), mouse anti-smooth muscle actin (SMA; Cell Signaling Technology), Lectin from tomato, FITC conjugate (Sigma), rabbit anti-BrdU (Abcam), and rabbit anti-GATA binding protein 4 (GATA4; Proteintech).
Fitc conjugate
Manufactured by Merck Group
Sourced in United States
FITC conjugate is a fluorescent labeling reagent used to attach fluorescein isothiocyanate (FITC) to various biomolecules, such as proteins, peptides, and nucleic acids. FITC is a widely used fluorescent dye that emits green fluorescence when excited by blue or ultraviolet light. The FITC conjugate can be used in various applications, including flow cytometry, fluorescence microscopy, and immunoassays, to detect and visualize labeled biomolecules.
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Lab products found in correlation
Rabbit anti brdu, by Abcam (1 mentions)
Alexa fluo series of second antibodies, by Thermo Fisher Scientific (1 mentions)
Rabbit anti anp, by Abcam (1 mentions)
Tissue tek oct compound, by Sakura Finetek (1 mentions)
Crest coated slides, by Matsunami (1 mentions)
Vectashield mounting medium containing dapi, by Vector Laboratories (1 mentions)
Lsm 710, by Zeiss (1 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa, by BD (1 mentions)
Fetal calf serum (fcs), by Merck Group (1 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (1 mentions)
Axio imager m2, by Zeiss (1 mentions)
Neuronal nuclei (neun), by Merck Group (1 mentions)
Blocking buffer, by Beyotime (1 mentions)
4 6 diamino 2 phenyl indole dapi solution, by Beyotime (1 mentions)
Cy3 conjugated goat anti rabbit, by Merck Group (1 mentions)
7 protocols using fitc conjugate
1
Multicolor Immunofluorescence Staining Protocol
2
Histological and Immunostaining Procedures for Reproductive Organs
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For hematoxylin and eosin staining, testes, epididymis and ovaries were fixed in 10% formalin or Bouin solution and embedded in paraffin. Sections were prepared on CREST-coated slides (Matsunami) at 6 μm thickness. The slides were dehydrated and stained with hematoxylin and eosin.
For Immunofluorescence staining, testes were embedded in Tissue-Tek O.C.T. compound (Sakura Finetek) and frozen. Cryosections were prepared on the CREST-coated slides (Matsunami) at 8 μm thickness and then air-dried and fixed in 4% paraformaldehyde in PBS at pH 7.4. The serial sections of frozen testes were fixed in 4% PFA for 5 min at room temperature and permeabilized in 0.1% TritonX100 in PBS for 10 min. The sections were blocked in 3% BSA/PBS or Blocking One (Nakarai), and incubated at room temperature with the primary antibodies in a blocking solution. After three washes in PBS, the sections were incubated for 1 h at room temperature with Alexa-dye-conjugated secondary antibodies in a blocking solution. PNA lectin staining was performed using Lectin from Arachis hypogaea, FITC conjugate (1:1000, Sigma-Aldrich L7381). TUNEL assay was performed using MEBSTAIN Apoptosis TUNEL Kit Direct (MBL 8445). DNA was counterstained with Vectashield mounting medium containing DAPI (Vector Laboratory). Statistical analyzes, and production of graphs and plots were done using GraphPad Prism9 or Microsoft Excel.
For Immunofluorescence staining, testes were embedded in Tissue-Tek O.C.T. compound (Sakura Finetek) and frozen. Cryosections were prepared on the CREST-coated slides (Matsunami) at 8 μm thickness and then air-dried and fixed in 4% paraformaldehyde in PBS at pH 7.4. The serial sections of frozen testes were fixed in 4% PFA for 5 min at room temperature and permeabilized in 0.1% TritonX100 in PBS for 10 min. The sections were blocked in 3% BSA/PBS or Blocking One (Nakarai), and incubated at room temperature with the primary antibodies in a blocking solution. After three washes in PBS, the sections were incubated for 1 h at room temperature with Alexa-dye-conjugated secondary antibodies in a blocking solution. PNA lectin staining was performed using Lectin from Arachis hypogaea, FITC conjugate (1:1000, Sigma-Aldrich L7381). TUNEL assay was performed using MEBSTAIN Apoptosis TUNEL Kit Direct (MBL 8445). DNA was counterstained with Vectashield mounting medium containing DAPI (Vector Laboratory). Statistical analyzes, and production of graphs and plots were done using GraphPad Prism9 or Microsoft Excel.
3
Immunostaining Tubulin Isoforms in Drosophila Embryos
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Mab YL1,2 against tyrosinated tubulin (Millipore) was used at 1∶2000. Anti-αTub67C rabbit polyclonal antibodies were made against a peptide from amino acids (aa) 35–61 of αTub67C, affinity-purified and used at 1∶400. Anti-αTub84B+D guinea pig polyclonal antibodies were made against the last 15 aa including the tyrosine residue of the common COOH-termini of αTub84B and αTub84D, respectively, and used as crude serum at 1∶300. DM1A was used as a FITC-conjugate (Sigma) at 1∶50 sequentially to mab YL1,2 in Fig. 1P . Polyclonal goat antibodies against Ncd (dS-17) were purchased from Santa Cruz Biotechnology and used at 1∶80. Polyclonal rabbit anti γTubulin and Msps antibodies were obtained from Y. Zheng and H. Ohkura, respectively, and were used both at 1∶1000. For actin staining, we hand-devitellinized the embryos and used Phalloidin, coupled to Alexa 488 (Invitrogen) at 1∶60. For YL1,2, we preferentially used 2nd antibodies coupled to 594 nm fluorochromes to obtain an optimal signal-to-noise ratio. All pictures were recorded on a Zeiss LSM 710.
4
Tomato Lectin-Based Flow Cytometry
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Approximately 1 million cells for each condition were harvested and washed twice with PBS before resuspension in PBS. Lectin from Lycopersicon esculentum (tomato) FITC conjugate (Sigma, L0401) was added in a 1:100 dilution before incubation for 1 h at 4 °C, protected from light. Cells were then washed twice with PBS, resuspended in PBS before flow cytometry analysis using LSR Fortessa (BD Biosciences) instruments. Downstream analysis was performed using FlowJo version 10.6.1 (BD Biosciences). Example FACS gating strategies are available in Supplementary Information.
5
Desialylation Inhibition by Anti-NanA Antibodies
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Raji cells (ATCC® CCL86™) used at 2.5 to 5 × 105 cells/well were incubated with recombinant NanA (1 nM) at 4°C for 30 min or infected with S. pneumoniae D39Δply at a multiplicity of infection (MOI) 10 for 3 h at 37°C in RPMI (Gibco, Thermo Fisher Scientific) + 10% FCS (Sigma). Inhibition of cell surface desialylation by α-NanA mAbs (4 – 1000 nM) was measured by flow cytometry (BD Accuri) using anti-asialo-GM1 polyclonal rabbit IgG (eBioscience) and Alexa Fluor® 488 F(ab’)2 fragment of goat anti-rabbit IgG H + L (Molecular Probes) as secondary reagent, or an A. hypogaea (peanut) lectin FITC conjugate (Sigma).
6
Immunohistochemical Analysis of Tissue Sections
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The cells were fixed with 4% PFA for 30 minutes, then the cells/tissue sections were washed with PBS for 5 minutes three times at room temperature. The frozen tissue sections were treated with formaldehyde at 0, 3, 6, 12 hours, 1, 3 and 7 days after modeling, then blocked with blocking buffer (Beyotime, Shanghai, China) for 1 hour. Next, the tissue sections were treated with primary antibodies [Ezh2, 1:200, rabbit monoclonal antibody (CST, USA); NF160/200, 1:500, mouse Monoclonal antibody (Sigma-Aldrich); IB4, 1:200, FITC conjugate (Sigma-Aldrich); TuJ1, 1:1 000, mouse Monoclonal antibody (Sigma-Aldrich); NeuN, 1: 300, rabbit monoclonal antibody] at 4°C overnight. Eighteen hours later, tissue sections were incubated with secondary antibodies [Cy3-conjugated goat anti-rabbit, 1:500 (Sigma); 488-conjugated goat anti-mouse, 1:500 (Sigma-Aldrich)] for 1.5 hours at room temperature, and then mounted on microscope slides. Finally, an antifade mounting medium with 4,6-diamino-2-phenyl indole (DAPI) solution (Beyotime) was used to seal the slice. Samples were visualized and images captured using an optical and epi-fluorescence microscope (Axio Imager M2, Carl Zeiss Microscopy GmbH, Jena, Germany). All assays were performed in triplicate.
7
Asaia Bacteria Expressing WSP in Mosquitoes
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Recombinant Asaia bacteria expressing WSP or with plasmid alone were grown as reported above; 10 μL of a cell suspension at the concentration of 108 cells ml−1 in PBS were placed on glass slides, air dried, and fixed for 20 min with cold methanol. Bacterial cells were blocked in bovine serum albumin (FBS) and probed with the primary goat anti-E tag antibody (Novus Biologicals), followed by incubation with an anti-goat IgG secondary antibody, FITC Conjugate (Sigma-Aldrich).
As for the detection of transgenic bacteria in mosquito organs, AsaiaWSP or AsaiapHM4 were administered to 2–3-day-old Ae. aegypti (Liverpool black-eyed strain) females via sugar meal (1 × 108 cells ml−1) plus kanamycin (100 μg ml−1) for 24 h; 24 h after the bacterial meal, fed mosquitoes were selected and their organs (crop, midgut and ovaries) dissected and fixed in 4% (wt vol−1) paraformaldehyde at 4 °C, washed in PBS, and blocked with 4% (wt vol−1) FBS. The samples were then probed with goat anti-E tag antibody, followed by incubation with a FITC-anti-goat IgG secondary antibody. Observations were recorded with a Leica microscope (LeicaTCSNT) and analyzed with ImageJ software. Survival of mosquitoes was also monitored daily. Survival percentages represent the mean survival percentage of three biological replicates of 30 mosquitoes each.
As for the detection of transgenic bacteria in mosquito organs, AsaiaWSP or AsaiapHM4 were administered to 2–3-day-old Ae. aegypti (Liverpool black-eyed strain) females via sugar meal (1 × 108 cells ml−1) plus kanamycin (100 μg ml−1) for 24 h; 24 h after the bacterial meal, fed mosquitoes were selected and their organs (crop, midgut and ovaries) dissected and fixed in 4% (wt vol−1) paraformaldehyde at 4 °C, washed in PBS, and blocked with 4% (wt vol−1) FBS. The samples were then probed with goat anti-E tag antibody, followed by incubation with a FITC-anti-goat IgG secondary antibody. Observations were recorded with a Leica microscope (LeicaTCSNT) and analyzed with ImageJ software. Survival of mosquitoes was also monitored daily. Survival percentages represent the mean survival percentage of three biological replicates of 30 mosquitoes each.
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