Purified R. akari cells on a coverslip were fixed and permeabilized as described earlier [67 (link)]. Rickettsial cells were washed three times in PBS (pH 7.2) containing 2% bovine serum albumin (BSA; mixtures, PBSA) and then blocked with 5% BSA in PBS for 1 h at 37 °C. After washing, bacterial cells were incubated with a “pre-absorbed” mouse anti-serum against 44 kDa hypothetical protein (1:100) diluted in 2% PBSA for 1 h at 37 °C. After washing, cells were incubated with goat anti-mouse IgG secondary antibody conjugated with Alexa fluor 488 (Life Technologies, USA) diluted 1:1000 in PBS containing 2% PBSA. The cells were washed three times with PBSA and blocked again with 5% BSA in PBS for 1 h at 37 °C. R. akari cells were then stained by using polyclonal rabbit antiserum against live R. akari diluted 1:200 and a goat anti-rabbit IgG conjugated with Rhodamine (Life technologies, USA) diluted 1:2000. After five times washing with PBS, the coverslip was dried and mounted with Vectashield (Vector Laboratories) and viewed with fluorescence microscopy (model Eclipse Ni, Nikon Japan).
Goat anti mouse igg secondary antibody conjugated with alexa fluor 488
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
Goat anti-mouse IgG secondary antibody conjugated with Alexa Fluor 488 is a laboratory reagent used for the detection and quantification of mouse immunoglobulin G (IgG) in various immunoassays. The Alexa Fluor 488 dye attached to the secondary antibody emits green fluorescence upon excitation, allowing for sensitive and specific detection of the target analyte.
Automatically generated - may contain errors
2 protocols using goat anti mouse igg secondary antibody conjugated with alexa fluor 488
1
Immunofluorescent Labeling of R. akari
2
Visualizing Sta1-3xHA Localization in Fungal Cells
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To visualize the localization of Sta1-3xHA protein on the surface of fungal cells, immunostaining was performed as described previously (Ma et al., 2018) . Ustilago maydis strains constitutively expressing Sta1-3xHA or Tin2-3xHA were suspended in 2% YEPSL containing 0.1 mM 16-hydroxyhexadecanoic acid at OD 600 = 0.5 and sprayed onto parafilm to induce filamentation (Mendoza-Mendoza et al., 2009) . The parafilm was incubated at 28°C for 16 h. After washing with phosphate-buffered saline (PBS), parafilm was incubated in PBS containing mouse anti-HA antibody (Sigma-Aldrich; 1 : 1500 dilution) and 3% bovine serum albumin at 4°C overnight. After washing, parafilm was incubated in PBS containing goat anti-mouse IgG secondary antibody conjugated with Alexa Fluor 488 (Life Technologies, Darmstadt, Germany; 1 : 1500 dilution) for 1 h at room temperature. After washing, the samples were analyzed using a TCS-SP8 confocal laser-scanning microscope (Leica Microsystems).
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