PWR-1E cells were washed in Dulbecco’s PBS (Lifetechnology, Monza, Italy), fixed in 4% paraformaldehyde in PBS for 10 min at RT, and permeabilized in PBS 0.1 M added with 0.1% Triton X-100 (Sigma, Milan, Italy) for 5 min. After washing in PBS at RT, cells were blocked with 10% Normal Donkey Serum (Jackson ImmunoResearch, West Grove, PA, USA) in PBS 0.1 M and incubated overnight at 4° C with the anti-human CNTF (1:100) and CNTFRɑ (1:150) antibodies (Table 1 ). Cells were then washed three times in PBS and incubated with the FITC-conjugated donkey anti-rabbit (for CNTF) and TRITC-conjugated anti-mouse (for CNTFRɑ) IgG secondary antibodies (both from Jackson ImmunoResearch) for 30 min at RT. TOTO3 probe was used for nuclear staining. Finally, the slides were cover-slipped with propyl gallate and evaluated with a Leica TCS-SL spectral confocal microscope.
Fitc conjugated donkey anti rabbit for cntf
Manufactured by Jackson ImmunoResearch
Sourced in United States
FITC-conjugated donkey anti-rabbit is a secondary antibody used for the detection of rabbit primary antibodies in immunoassays. The antibody is conjugated with the fluorescent dye FITC (Fluorescein isothiocyanate) which allows for the visualization of the target protein.
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2 protocols using fitc conjugated donkey anti rabbit for cntf
1
Immunocytochemical Analysis of CNTF and CNTFRα
2
Immunofluorescence Staining of LNCaP and 22Rv1 Cells
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Immunofluorescence staining was performed as previously described [24 (link),25 (link)]. Briefly, LNCaP and 22Rv1 cells were washed in Dulbecco’s PBS (Life technology, Monza, Italy), fixed in 4% paraformaldehyde in PBS for 10 min at room temperature (RT), and permeabilized in PBS 0.1 M added with 0.1% Triton X-100 (Sigma, Milano, Italy) for 5 min. After washing in PBS at RT, cells were blocked with 10% Normal Donkey Serum (Jackson ImmunoResearch, Pennsylvania, PA, USA) in PBS 0.1 M and incubated overnight at 4 ℃ with the primary antibodies listed in Table 1 . Cells were then washed 3 times in PBS and incubated with the FITC-conjugated donkey anti-rabbit (for CNTF) and TRITC-conjugated anti-mouse (for CNTFRα) IgG secondary antibodies (both from Jackson ImmunoResearch, West Grove, PA, USA) 1:400 for 30 min at room temperature. TOTO3 (1:5000) probe was used for nuclear staining. Finally, the slides were cover-slipped with propyl gallate and evaluated with a Leica TCS-SL spectral confocal microscope. For negative controls, see the immunohistochemistry Section 2.2 .
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