Syntaxin 10 siRNA or non-targeting control siRNA transfected monolayers were infected with C. trachomatis serovar L2 for 36 h. Infected monolayers were collected and fixed in 2% EM-grade paraformaldehyde plus 2.5% EM-grade glutaraldehyde (Polysciences Inc., Warrington, PA) in 100 mM sodium phosphate buffer (Sigma Aldrich). Cells were processed for TEM as described previously (Beatty, 2006 (link)). TEM images were taken for two independent experiments. TEM images were used to quantify total numbers of organisms per inclusion, percentage of developmental forms in each inclusion and inclusion diameter. The diameter measurement was taken at the widest part of each inclusion. The results are displayed as μm units, based on the scale set from the electron microscope. All means and standard error of the means were calculated using GraphPad Prism 6 software.
Em grade paraformaldehyde
Manufactured by Polysciences
EM-grade paraformaldehyde is a chemical compound used as a fixative in electron microscopy (EM) sample preparation. It is a solid, white, crystalline material that is highly reactive and forms a stabilized solution when mixed with water or other solvents. The primary function of EM-grade paraformaldehyde is to preserve the structure and morphology of biological samples for observation and analysis under an electron microscope.
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Lab products found in correlation
Goat anti gfp, by Abcam (5 mentions)
Rabbit anti lacz, by MP Biomedicals (4 mentions)
Draq5, by Cell Signaling Technology (3 mentions)
Rabbit and mouse anti ph3, by Merck Group (3 mentions)
Lsm image browser, by Zeiss (2 mentions)
Immersol 518f, by Zeiss (2 mentions)
Alexa fluor conjugated secondary antibody, by Jackson ImmunoResearch (2 mentions)
Lsm 800 confocal microscope, by Adobe (2 mentions)
Alexa fluor 546 donkey anti mouse and anti rabbit, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 633 donkey anti mouse and anti rabbit and 488 donkey anti goat, by Jackson ImmunoResearch (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Sodium phosphate buffer, by Merck Group (1 mentions)
Trypsin, by Sartorius (1 mentions)
Phloretin, by Merck Group (1 mentions)
Dynasore, by Merck Group (1 mentions)
Calphostin c, by Merck Group (1 mentions)
Sodium pyruvate, by Sartorius (1 mentions)
G418 antibiotic, by Thermo Fisher Scientific (1 mentions)
Filipin, by Merck Group (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
7 protocols using em grade paraformaldehyde
1
Quantification of Chlamydia Inclusion Dynamics
2
Endocytosis Inhibition in Cell Experiments
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Phenol red-free Dulbecco's modified eagle medium (DMEM), phosphate-buffered saline with Mg2+ and Ca2+ (PBS), mannitol, phorbol 12-myristate 13-acetate (PMA), phloretin, calphostin C, Filipin and Dynasore were purchased from Sigma Aldrich (St Louis, MO). Fetal Bovine Serum (FBS), l -alanine-l -glutamine, trypsin and sodium pyruvate were ordered from Biological Industries (Beit-Haemek, Israel). Lipofectamine 2000 and G418 antibiotic were purchased from Life Technologies (Carlsbad, CA). EM-grade paraformaldehyde was bought from Polysciences (Warrington, PA), Pitstop2 from ABCAM (Cambridge, UK) and conjugated human holo-transferrin from Jackson Laboratories (Sacramento, CA). Unless otherwise noted, all other reagents were ordered from Sigma Aldrich.
3
Drosophila Gastrointestinal Tract Immunostaining
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Female flies were used for gut immunostaining in all experiments. The entire gastrointestinal tracts were dissected out and fixed in 1× PBS plus 8% EM-grade paraformaldehyde (Polysciences) for 2 h. Samples were washed and incubated with primary and secondary antibodies in a solution containing 1× PBS, 0.5% BSA, and 0.1% Triton X-100. The following primary antibodies were used: rabbit anti-Gish (Tan et al., 2010 (link)), 1:500; mouse anti-Delta (Developmental Studies Hybridoma Bank), 1:100; rabbit anti-lacZ (Cell Signaling Technology), 1:1,000; mouse anti-pH3 (Millipore), 1:2,000; goat anti-GFP (Abcam), 1:500; rabbit anti–cleaved caspase-3, 5A1E (Cell Signaling Technology), 1:1,000; and DRAQ5 (Cell Signaling Technology), 1:5,000. Alexa Fluor–conjugated secondary antibodies were used at 1:400 (Jackson ImmunoResearch and Invitrogen). Guts were mounted in 70% glycerol and imaged with an inverted confocal microscope (LSM 710; Zeiss) using 10×, 20×, and 40× oil objectives (imaging medium: Immersol 518F; Zeiss). Imaging acquisition was performed at room temperature with LSM Image Browser (Zeiss), and image processing was done in Adobe Photoshop CC.
4
Gut Immunostaining in Female Flies
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Female flies were used for gut immunostaining in all experiments. The entire gastrointestinal tracts were dissected out and fixed in PBS plus 8% EM grade paraformaldehyde (Polysciences) for 2 h. Samples were washed and incubated with primary and secondary antibodies in a solution containing PBS, 0.5% BSA, and 0.1% Triton X-100. The following primary antibodies were used: mouse anti-Pros (Developmental Studies Hybridoma Bank), 1:100; rabbit anti-LacZ (MP Biomedicals), 1:1,000; rabbit and mouse anti-PH3 (EMD Millipore), 1:1,000; goat anti-GFP (Abcam), 1:1000; rabbit anti-Pdm1 (aa 95–256; gift from X. Yang, Institute of Molecular and Cell Biology, Singapore, Singapore); DRAQ5 (Cell Signaling Technology), 1:5,000; Hoechst (Life technologies), 1:500. Alexa Fluor–conjugated secondary antibodies were used at 1:400 (Jackson ImmunoResearch Laboratories, Inc., and Invitrogen). Guts were mounted in 70% glycerol and imaged with a confocal microscope (LSM 510 inverted; Carl Zeiss) using 10×, 20×, and 40× oil objectives (imaging medium: Immersol 518F; Carl Zeiss). The imaging temperature was room temperature. The acquisition and processing software was LSM Image Browser (Carl Zeiss) and image processing was done in Photoshop CC (Adobe).
5
Gut Immunostaining in Female Flies
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Female flies were used for gut immunostaining in all experiments. The entire intestine was dissected out and fixed in 1× PBS plus 8% EM‐grade paraformaldehyde (Polysciences) for 1 h. Samples were washed and incubated with primary and secondary antibodies in a solution containing 1× PBS, 0.5% BSA, and 0.1% Triton X‐100. The following primary antibodies were used: mouse anti‐Dl (DSHB), 1:100; rabbit anti‐LacZ (MP Biomedicals), 1:1,000; rabbit and mouse anti‐PH3 (Millipore), 1:1,000; goat anti‐GFP (Abcam), 1:1,000; Mouse anti‐Pros (MR1A); Rabbit anti‐Pdm1 (gift from X. Yang, Institute of Molecular and Cell Biology, Singapore). Secondary antibodies conjugated to Alexa Fluor 546 donkey anti‐mouse and anti‐rabbit (Molecular Probes) and Alexa Fluor 633 donkey anti‐mouse and anti‐rabbit and 488 Donkey anti‐goat (Jackson immunoresearch) were used at 1:400. Fluorescently labeled samples were counterstained with DAPI for visualization of DNA. Images were captured with a Zeiss LSM 800 confocal microscope and assembled in Adobe Photoshop.
6
Gut Immunostaining in Drosophila Females
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Female flies were used for gut immunostaining in all experiments. The entire gastrointestinal tracts were dissected out and fixed in 1 X PBS plus 8% EM grade paraformaldehyde (Polysciences) for 2 hr. Samples were washed and incubated with primary and secondary antibodies in a solution containing 1 X PBS, 0.5% BSA, and 0.1% Triton X-100. The following primary antibodies were used: mouse anti-Delta (DSHB), 1:100; mouse anti-Pros (DSHB), 1:100; mouse anti-Arm (DSHB), 1:100; mouse anti-integrin βPS/Mys (DSHB), 1:100; rabbit anti-LacZ (MP Biomedicals), 1:1000; rabbit and mouse anti-PH3 (Millipore, Billerica, MA), 1:1000; goat anti-GFP (Abcam, Cambridge, MA), 1:1000; mouse anti-pMad antibody (Persson et al., 1998 (link)), 1:300; rabbit anti-Pdm1 (gift from Dr Xiaohang Yang, Institute of Molecular and Cell Biology, Singapore), rabbit anti-Lava lamp, 1:300; 1:500; DRAQ5 (Cell Signaling Technology, Danvers, MA), 1:5000; Phalloidin, 1:100. Quantification of immunostaining was performed using ImageJ software.
7
Fly Gut Immunostaining Protocol
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Female flies were used for gut immunostaining in all experiments. The entire intestine was dissected out and fixed in 1× PBS plus 8% EM-grade paraformaldehyde (Polysciences) for 1 h.
Samples were washed and incubated with primary and secondary antibodies in a solution containing 1× PBS, 0.5% BSA, and 0.1% Triton X-100. The following primary antibodies were used: mouse anti-Delta (DSHB), 1:100; rabbit anti-LacZ (MP Biomedicals), 1:1,000; rabbit and mouse anti-PH3 (Millipore), 1:1,000; goat anti-GFP (Abcam), 1:1,000; Mouse anti-Pros (MR1A); Rabbit anti-Pdm1 (gift from X. Yang, Institute of Molecular and Cell Biology, Singapore).
Secondary antibodies conjugated to Alexa Fluor 546 donkey anti-mouse and anti-rabbit (Molecular Probes) and Alexa Fluor 633 donkey anti-mouse and anti-rabbit and 488 Donkey antigoat (Jackson immunoresearch) were used at 1:400. Fluorescently labeled samples were counterstained with DAPI for visualization of DNA. Images were captured with a Zeiss LSM 800 confocal microscope and assembled in Adobe Photoshop.
Samples were washed and incubated with primary and secondary antibodies in a solution containing 1× PBS, 0.5% BSA, and 0.1% Triton X-100. The following primary antibodies were used: mouse anti-Delta (DSHB), 1:100; rabbit anti-LacZ (MP Biomedicals), 1:1,000; rabbit and mouse anti-PH3 (Millipore), 1:1,000; goat anti-GFP (Abcam), 1:1,000; Mouse anti-Pros (MR1A); Rabbit anti-Pdm1 (gift from X. Yang, Institute of Molecular and Cell Biology, Singapore).
Secondary antibodies conjugated to Alexa Fluor 546 donkey anti-mouse and anti-rabbit (Molecular Probes) and Alexa Fluor 633 donkey anti-mouse and anti-rabbit and 488 Donkey antigoat (Jackson immunoresearch) were used at 1:400. Fluorescently labeled samples were counterstained with DAPI for visualization of DNA. Images were captured with a Zeiss LSM 800 confocal microscope and assembled in Adobe Photoshop.
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