mouse polyclonal antibody against gfp jl 8  (TaKaRa)

Journal: PLoS Pathogens

Article Title: Polarized Cell Division of Chlamydia trachomatis

doi: 10.1371/journal.ppat.1005822

Figure Lengend Snippet: HeLa cells infected with C . trachomatis were fixed at 2, 4, 8 (A and B), or 10 (C) hours post-infection (hpi). Following fixation, the cells were permeabilized and LPS localization was determined by incubating cells with anti-LPS mouse monoclonal antibodies followed by donkey anti-mouse IgG conjugated to Alexa Fluor 568 (A; red), Hsp60 localization was determined using anti-Hsp60 rabbit polyclonal antibodies followed by donkey anti-rabbit IgG conjugated to Alexa Fluor 633 (A, B, and C; blue), and MOMP localization was determined using anti-MOMP goat polyclonal antibodies followed by donkey anti-goat IgG conjugated to Alexa Fluor 488 (A, B, and C; green). Following the antibody incubations, the cells were imaged by confocal microscopy. (B and C) DNA was visualized by staining with Hoechst 33342 (red). White bars are 0.5μ.

Article Snippet: Mouse polyclonal antibodies against GFP were purchased from Zymed.

Techniques: Infection, Confocal Microscopy, Staining

Journal: PLoS Pathogens

Article Title: Polarized Cell Division of Chlamydia trachomatis

doi: 10.1371/journal.ppat.1005822

Figure Lengend Snippet: HeLa cells infected with C . trachomatis were fixed at 11 (A and C) or 13 (B) hours post-infection. Following permeabilization, the cells were incubated with rabbit polyclonal antibodies against Hsp60 (blue) and goat polyclonal antibodies against MOMP (green) followed by donkey anti-rabbit IgG conjugated to Alexa Fluor 568 and donkey anti-goat IgG conjugated to Alexa Fluor 488 secondary antibodies. The cells were then washed and stained with Hoechst 33342 prior to confocal analysis. Arrow in A points to a region of polarized MOMP in a mother cell. Dash in A denotes the presence of a MOMP-poor septum separating the mother and daughter cell. Arrowhead in A points to Hsp60 in the daughter cell. (B) Localization profile of MOMP, Hsp60, and DNA in a cell that has completed its first division. Arrow in B points to a MOMP-poor region in the center of the septum between the mother and daughter cell. (C) Examples of C . trachomatis at 11 hours post-infection that were simultaneously undergoing polar growth from two sites in the cell. (D) Infected HeLa cells were fixed at 10, 11, 12, or 13 hours post-infection and the percentage of C . trachomatis that were undergoing polarized cell division (black bars) or had completed the first division (white bars) were quantified. All of the MOMP and Hsp60 double-positive cells in randomly selected fields were included in the analysis. White bars are 0.5μ.

Article Snippet: Mouse polyclonal antibodies against GFP were purchased from Zymed.

Techniques: Infection, Incubation, Staining

Journal: PLoS Pathogens

Article Title: Polarized Cell Division of Chlamydia trachomatis

doi: 10.1371/journal.ppat.1005822

Figure Lengend Snippet: (A) HeLa cells infected with C . trachomatis were incubated in the presence of green fluorescent BODIPY FL C5 ceramide as described in the Materials and Methods and polarized cell division intermediates were imaged in live cells at 11 hours post-infection using a Zeiss AxioImager.M2 microscope. (B) HeLa cells were infected with C . trachomatis serovar L2 that contained an anhydrotetracycline (aTc)-inducible plasmid expressing GFP-FtsQ . aTc was added to infected cultures at 8 hours post-infection then the cells were labeled with red fluorescent BODIPY TR C5 ceramide. Polarized cell division intermediates were imaged in live cells at 11 hours post-infection using a Zeiss LSM710 confocal microscope. Arrowheads in A and B indicate regions of intense BODIPY-sphingomyelin fluorescence at one pole of a round cell prior to division. The polar patch of sphingomyelin fluorescence in B also contained GFP-FtsQ. Arrows in A and B indicate a sphingomyelin-rich membrane separating a nascent daughter cell from a mother cell. (C) HeLa cells were infected with C . trachomatis serovar L2 that contained aTc-inducible version of GFP-FtsQ. The fusion was induced by the addition of aTc to cultures at 8 hours post-infection and the cells were fixed at 11 hours post-infection. Following permeabilization, the cells were incubated with rabbit polyclonal antibodies against Hsp60 (blue), goat polyclonal antibodies against MOMP (red), and mouse monoclonal antibodies against GFP (green) followed by donkey anti-rabbit IgG conjugated to Alexa Fluor 633, donkey anti-goat IgG conjugated to Alexa Fluor 594, and donkey anti-mouse IgG conjugated to Alexa Fluor 488 secondary antibodies. The cells were then washed prior to imaging on a Zeiss AxioImager.M2 microscope. Note the size and morphology of (B) live and (C) fixed cells are very similar. Arrowhead in C indicates a region of GFP-FtsQ fluorescence in the MOMP-positive pole of the cell prior to cell division.

Article Snippet: Mouse polyclonal antibodies against GFP were purchased from Zymed.

Techniques: Infection, Incubation, Microscopy, Plasmid Preparation, Expressing, Labeling, Fluorescence, Imaging

Journal: PLoS Pathogens

Article Title: Polarized Cell Division of Chlamydia trachomatis

doi: 10.1371/journal.ppat.1005822

Figure Lengend Snippet: (A) HeLa cells were infected with C . trachomatis and fixed at 13 hours post-infection. Following permeabilization, the cells were incubated with rabbit polyclonal antibodies against Hsp60 (blue) and goat polyclonal antibodies against MOMP (green) followed by goat anti-rabbit IgG conjugated to Alexa Fluor 633 and donkey anti-goat IgG conjugated to Alexa Fluor 488 secondary antibodies. The cells were then washed prior to confocal analysis. Arrowheads in A point to sites of polarized growth in cells undergoing the second round of division. (B and C) Alternatively, cells were fixed at 17 hours post-infection and processed for transmission electron microscopy. Black arrows in B and C point to cells undergoing polarized cell division. (D) Model depicting the morphological changes that occur during the primary differentiation and the initial cell division of C . trachomatis are illustrated. The localization of MOMP (green), Hsp60 (blue), and sphingomyelin (red) is shown. The distribution of sphingomyelin has not been determined in the two earliest intermediates in this model (left hand side of D). The chlamydial membrane is depicted by a dashed line in these intermediates. White bar in A is 0.5μ. The black bars in B and C are 1μ.

Article Snippet: Mouse polyclonal antibodies against GFP were purchased from Zymed.

Techniques: Infection, Incubation, Transmission Assay, Electron Microscopy

Journal: PLoS Pathogens

Article Title: Polarized Cell Division of Chlamydia trachomatis

doi: 10.1371/journal.ppat.1005822

Figure Lengend Snippet: HeLa cells were infected with C. trachomatis and fixed at 14 hours post-infection. The cells were then permeabilized and incubated with anti-MOMP goat polyclonal antibodies (green) and anti-IncG rabbit polyclonal antibodies (red) followed by donkey anti-goat IgG conjugated to Alexa Fluor 488 and donkey anti-rabbit IgG conjugated to Alexa Fluor 568 secondary antibodies (A and B). The cells were then washed and stained with Hoechst 33342 (blue) prior to confocal analysis. The images in B are consecutive confocal slices from a z-stack. Arrowheads in A and B point to sites of asymmetric membrane expansion. Asterisk in B indicates a cell simultaneously undergoing polar growth from two sites. The white bars are 0.5μ. The numbers in the merged images in B correspond to the position in the Z-stack in microns.

Article Snippet: Mouse polyclonal antibodies against GFP were purchased from Zymed.

Techniques: Infection, Incubation, Staining

Journal: PLoS Pathogens

Article Title: Polarized Cell Division of Chlamydia trachomatis

doi: 10.1371/journal.ppat.1005822

Figure Lengend Snippet: (A, B, and D) HeLa cells were infected with C. trachomatis serovar L2 and fixed at 11 hours post-infection. The cells were then permeabilized and LPS localization was determined by incubating cells with anti-LPS mouse monoclonal antibodies (A; red), RNA polymerase β subunit localization was determined using mouse monoclonal antibodies (Pol. in D; red), EF-Tu localization was determined using anti-EF-Tu mouse polyclonal antibodies (B; red). The distribution of the mouse primary antibodies was visualized by incubating cells with donkey anti-mouse IgG conjugated to Alexa Fluor 568. Hsp60 localization was visualized using anti-Hsp60 rabbit polyclonal antibodies followed by donkey anti-rabbit IgG conjugated to Alexa Fluor 633 (A and B—blue; D—cyan) and MOMP localization was visualized using anti-MOMP goat polyclonal antibodies followed by donkey anti-goat IgG conjugated to Alexa Fluor 488 (A, B, and D; green). In some instances, the cells were stained with Hoechst (D; blue) prior to confocal analysis. Each panel contains various intermediates in the polarized cell division process. Arrowheads in D denote regions in a nascent daughter cell where the DNA and the RNA polymerase β subunit co-localize. Asterisks in D mark nascent daughter cells that contain DNA but lack the RNA polymerase β subunit and Hsp60. Arrows in D point to region where the RNA polymerase β subunit and the bacterial chromosome co-localize in a nascent daughter cell that does not yet contain Hsp60. In Panel D, Merge1: merge of MOMP/ RNA polymerase β subunit/Hsp60; Merge2: merge of MOMP/ RNA polymerase β subunit/DNA. White bars in A, B, and D are 0.5μ. (C) A lysate prepared from HeLa cells infected with C . trachomatis at 24 hours post-infection was subjected to immunoblotting analysis with the EF-Tu antibodies. A single 43kDa species that matches the predicted molecular mass of EF-Tu was observed.

Article Snippet: Mouse polyclonal antibodies against GFP were purchased from Zymed.

Techniques: Infection, Staining, Western Blot