Murine splenic NK cells were MACS enriched, let adhere on poly-D-Lysine (Sigma-Aldrich) coated coverslips, fixed with 4% PFA, permeabilized with 0.1% Triton X-100, incubated with blocking buffer (5% normal donkey serum (Sigma-Aldrich), 2% BSA, 0.05% Tween). Cells were then stained with biotin-conjugated goat polyclonal anti-SIGIRR antibody or biotin-conjugated normal goat IgG as control (both R&D Systems) (10µg/ml) followed by Alexa Fluor 488–conjugated donkey anti-goat IgG antibody (Molecular Probes) and DAPI (Invitrogen). Coverslips were mounted with the antifade medium FluorPreserve Reagent (EMD Millipore) and analyzed with an Olympus Fluoview FV1000 laser scanning confocal microscope with oil immersion lens 40× (N.A.1.3).
Fluorpreserve reagent
Manufactured by Merck Group
FluorPreserve Reagent is a liquid-based preservative designed for the stabilization of fluorescent-labeled biomolecules. It helps maintain the fluorescent signal intensity of labeled proteins, nucleic acids, and other biological samples during storage and handling.
Automatically generated - may contain errors
Lab products found in correlation
Normal donkey serum (nds), by Merck Group (2 mentions)
Biotin conjugated goat polyclonal anti sigirr antibody, by R&D Systems (2 mentions)
Biotin conjugated normal goat igg, by R&D Systems (2 mentions)
Alexa fluor 488 conjugated donkey anti goat igg antibody, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Poly d lysine, by Merck Group (2 mentions)
Fluoview fv1000 confocal laser scanning microscope, by Olympus (2 mentions)
Lsm800 confocal microscope, by Zeiss (2 mentions)
Dapi 4 6 diamidino 2 phenylindole, by Vector Laboratories (1 mentions)
Bz 9000 e hs all in one fluorescence microscope, by Keyence (1 mentions)
3 protocols using fluorpreserve reagent
1
Immunofluorescence Analysis of SIGIRR in Murine Splenic NK Cells
2
Immunofluorescence Analysis of SIGIRR in Murine Splenic NK Cells
3
Immunofluorescence Staining of NRVCMs
Check if the same lab product or an alternative is used in the 5 most similar protocols
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NRVCMs and mouse tissue cryosections were fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. After permeabilization and blocking with 2.5% BSA and 0.1% Triton X-100 in PBS for 1 h at room temperature, the primary antibody was applied and the slides were incubated at 4°C overnight. Flourescent-dye conjugated secondary antibodies were used at a dilution of 1:500 for 1 h at room temperature (Supplementary Table S1 ). Nuclei staining was performed simultaneously with DAPI (4′,6′-diamidino-2-phenylindole, Vector Laboratories). Finally, slides were mounted with FluorPreserve reagent (Merck). Images were captured on a BZ-9000-E HS all-in-one fluorescence microscope (Keyence) or a Zeiss LSM-800 confocal microscope (Axio Observer. Z1/7 microscope).
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