For immuno-staining, tissue sections were first washed in 0.01 M PBS (3 × 10 min) and then incubated in 0.01 M PBS containing 10% normal goat serum (NGS), 0.1% Triton X-100, and 3% bovine serum albumin for 1 h to decrease non-specific staining. The sections were then incubated for 24 h at 4°C in 0.01 M PBS containing anti-ΔFosB (SC-48; 1:400; Santa Cruz) or anti-pCREB rabbit polyclonal antibody (06–519; 1:400; Millipore). Unbound primary antibodies were washed in 0.01 M PBS (3 × 10 min) prior to 24 h incubation at 4°C with FITC-conjugated goat anti-rabbit secondary antibody (1:400; Sigma-Aldrich) followed rinse in PBS for 3 × 10 min. Finally, 4′, 6′-diamidino-2-phenylindole (DAPI) was added to label nuclei, and the sections were mounted with mounting medium for fluorescence observation.
Fitc conjugated goat anti rabbit secondary antibody
Manufactured by Merck Group
Sourced in United States
The FITC-conjugated goat anti-rabbit secondary antibody is a laboratory reagent used to detect the presence of rabbit primary antibodies in various biological assays. It is labeled with the fluorescent dye FITC (Fluorescein Isothiocyanate) to enable visualization of the target analyte.
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Lab products found in correlation
Normal horse or goat serum, by Vector Laboratories (1 mentions)
Anti vimentin, by Proteintech (1 mentions)
Goat anti snail, by Abcam (1 mentions)
Rabbit anti phh3, by Merck Group (1 mentions)
Biotinylated species specific secondary antibodies, by Vector Laboratories (1 mentions)
Cy3 conjugated streptavidin, by GE Healthcare (1 mentions)
Bovine serum albumin (bsa), by Vector Laboratories (1 mentions)
Slowfade gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Carl lsm710, by Zeiss (1 mentions)
Alexafluor 546 goat anti mouse secondary antibody, by Thermo Fisher Scientific (1 mentions)
Eclipse ti, by Nikon (1 mentions)
Lab tek chamber slide system, by Thermo Fisher Scientific (1 mentions)
Anti laminin, by Merck Group (1 mentions)
Anti fibronectin, by Merck Group (1 mentions)
Eclipse 50i, by Nikon (1 mentions)
4 protocols using fitc conjugated goat anti rabbit secondary antibody
1
Immunostaining Protocol for ΔFosB and pCREB
2
Immunofluorescence Staining of E14.5 Mouse Hearts
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E14.5 embryonic hearts were dissected into ice cold PBS, embedded in OCT (R.A. Lamb) and snap frozen in liquid nitrogen. Cryosections (14 μm thick) were fixed in 4% PFA for 15 minutes, permeabilised in PBS + 0.1% (v/v) Triton X-100 for 15 minutes and subsequently blocked with PBS + 1% (w/v) BSA + 10% (v/v) normal goat or horse serum (Vector) for 1 hour. Sections were then incubated with the following primary antibodies diluted in PBS + 0.1% (v/v) Triton X-100 for 24 hours at 4°C: rabbit polyclonal anti-mouse Wt-1 (Calbiochem, CA1026, 1:300), goat anti-Snail (Abcam, ab53519, 1:100), rabbit anti-PHH3 (Millipore, 06–570, 1:300), anti-vimentin (Proteintech, 10366-1-AP, 1:50). Sections were then incubated with species-specific biotinylated secondary antibodies (Vector) at 1:500, for 2 hours, or FITC conjugated goat anti-rabbit secondary antibody (Sigma, F9887, 1:160) (detection of PHH3). Sections were incubated with Cy3 conjugated streptavidin (GE Healthcare, PA43001), diluted 1:3000 or 1:1000 (detection of Snail) for 30 minutes. Coverslips were mounted with Vectashield with DAPI (Vector, H-1200) and sealed with nail varnish. Slides were stored in the dark at 4°C and imaged within 48 hours.
3
Quantifying NF-κB Activation in Cell Lines
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Transduced HONE1 and HK1 cell lines were fixed in formalin for 10 min and permeabilized with 0.5% Triton X-100 in PBS as described [33 (link)]. The phosphorylated p65 Serine 536 primary antibody (1:100) was incubated with the cells overnight at 4°C, followed by a FITC-conjugated goat anti-rabbit secondary antibody (Sigma-Aldrich, St. Louis, MO, USA) (1:100). Total IκBa was labeled with Alexa Fluor 546 goat anti-mouse secondary antibody (Molecular Probes, Life Technology, Carlsbad, CA, USA). Nuclei were stained with DAPI, cells were mounted onto glass slides with Slowfade Gold Antifade Reagent (Molecular Probes) and viewed under a fluorescence inverted microscope (Nikon Eclipse Ti, Nikon Instruments, Melville, NY, USA) or confocal microscope (Carl Zeiss LSM 710, Zeiss Microscopy, Jena, Thuringia, Germany).
4
Immunofluorescence Analysis of 2BH4 Cell Extracellular Matrix
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2BH4 cells (2 × 104 cells) were plated in an 8-well Lab-Tek Chamber Slide™ System (Nunc, NY, USA) for 16 h, treated with PRL for 24 h, washed in PBS, fixed with methanol (Synth, Diadema, SP, Brazil) for 10 min, and subjected to an indirect immunofluorescence assay. TECs were pre-incubated for 30 min with PBS containing 1% BSA, followed by the addition of primary antibodies such as anti-fibronectin, anti-laminin (both from Sigma-Aldrich), and anti-CXCL12 (eBioscience) for 1 h in a humid chamber at room temperature (25°C). Next, the cells were washed three times with PBS and incubated for 45 min with FITC-conjugated goat anti-rabbit secondary antibody (Sigma-Aldrich). Subsequently, the cells were washed again in PBS, and the slides were mounted in glycerol/PBS (1:3). As a negative control, the cells were not incubated with the primary antibody, but only with the secondary antibody, so as not to produce any signal. After covering with a glass coverslip, the slides were examined using fluorescence microscopy (Nikon Eclipse 50i; Nikon Instruments Inc., Chicago, IL, USA). The fluorescence intensity was determined in pixels and quantified using Image J version 1.44 (NIH, MD, USA).
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