Brains were extracted following transcardial perfusion with saline followed by 10% formalin at room temperature and embedded in paraffin. Hippocampal lesions were generated in 5 μm coronal sections by transverse incision of SC fibers. Deparaffinized sections were permeabilized with 0.1% Triton X-100 in PBS and blocked in 0.1% Saponin and 3% bovine serum albumin in PBS (PBSS). Sections were incubated in Anti-NeuN Alexa Fluor®488 conjugated antibody (EMD Millipore MAB377X 1:250) in PBSS, washed and mounted with ProLong™ Diamond containing DAPI (ThermoFisher Scientific). Tile-scanned 20× images were collected using a Zeiss LSM 700 confocal microscope (Carl Zeiss Inc., Thornwood, NY).
Prolong diamond containing dapi
Manufactured by Thermo Fisher Scientific
ProLong™ Diamond containing DAPI is a mounting medium used for the preservation and visualization of fluorescently labeled biological samples. It is designed to maintain the brightness and stability of fluorescent signals while counterstaining nuclei with the DNA-binding dye DAPI.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 700 confocal microscope, by Zeiss (1 mentions)
Lsm 700 laser scan confocal microscope, by Zeiss (1 mentions)
Alexa fluor 594, by Jackson ImmunoResearch (1 mentions)
Lysotracker red dnd 99, by Thermo Fisher Scientific (1 mentions)
Phosphorylated stat3, by Cell Signaling Technology (1 mentions)
Dq red bsa, by Thermo Fisher Scientific (1 mentions)
Lamp1, by Developmental Studies Hybridoma Bank (1 mentions)
4 protocols using prolong diamond containing dapi
1
Formalin-Fixed Hippocampal Lesion Analysis
2
Quadruple FISH Probe Protocol
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Fluorescence in situ hybridization analyses were performed as previously described70 (link) using ZytoLight SPEC p16/CEN3/7/17 Quadruple Color Probes (Zytovision). Briefly, cells on coverslips were fixed in Carnoy's fixative solution (75% methanol and 25% acetic acid), washed in 2 × SSC (1 × SSC is 0.15 M NaCl and 0.015 M sodium citrate) for 2 min and then dehydrated in an ethanol series (70, 85 and 100%; 2 min each following air drying). The DNA probes were pre-warmed at 37 °C, placed onto the coverslips and denatured at 75 °C for 2 min. The coverslips were then incubated overnight at 37 °C, washed with 4 × SSC containing 0.05% Tween 20 for 5 min and then incubated in 0.25 × SSC at 72 °C for 2 min. After a wash in 4 × SSC containing 0.05% Tween 20 for 30 s, the coverslips were mounted in ProLong Diamond containing DAPI (Thermo Fisher).
3
Immunofluorescence Staining of C. burnetii
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The experimental steps of immunofluorescence staining were described in detail elsewhere (Hayek et al., 2019 (link)). Briefly, macrophages were seeded on 10 mm sterile coverslips in 24-well plates. After infection and incubation, the cells were fixed with 4% paraformaldehyde (PFA) and permeabilized with ice-cold methanol. The cells were then quenched with 50 mM NH4Cl in PBS/5% goat serum (GS) followed by incubation with the primary antibody against C. burnetii NMII (Davids Biotechnology). Alexa Fluor 594 (Jackson ImmunoResearch Labs) was used as the secondary antibody. Finally, the slides were mounted with ProLong Diamond containing DAPI (Invitrogen). Immunofluorescent images were taken using the Carl Zeiss LSM 700 Laser Scan Confocal Microscope and the ZEN2009 software.
4
Immunofluorescence Microscopy of C. burnetii
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For indirect immunofluorescence microscopy analyses, C. burnetii infected MF were cultured on 10mm coverslips in 24-well dishes. At indicated points of time, cells were fixed for 15 min with 4% paraformaldehyde (PFA), permeabilized 1 min with ice-cold methanol and quenched and blocked for 30 min with 50 mM NH 4 Cl in PBS/ 5% goat serum (GS). After incubation with primary and secondary antibodies dilution in PBS/ 5% GS the coverslips were mounted using ProLong Diamond containing DAPI (Invitrogen). For visualization, a Carl Zeiss LSM 700 Laser Scan Confocal Microscope and the ZEN2009 software (Jena, Germany) were used.
In this study, we used primary antibodies directed against C. burnetii, LAMP-1 (Developmental Studies Hybridoma Bank, Iowa, IA, USA), and phosphorylated STAT3 (Cell Signaling, 9145). Secondary antibodies were Alexa Fluor labeled (Alexa Fluor 488 and 594) and purchased from Dianova, Hamburg, Germany. To detect acidic cell compartments, infected MF were incubated with equilibrated 1 mM LysoTracker Red DND-99 (Life Technologies) diluted in RPMI-CM one hour prior to fixation. As a phagolysosomal marker, DQ-Red BSA (Life Technologies) was used at a concentration of 2 mg/ml and incubated with the infected MF 4 hours prior to fixation.
In this study, we used primary antibodies directed against C. burnetii, LAMP-1 (Developmental Studies Hybridoma Bank, Iowa, IA, USA), and phosphorylated STAT3 (Cell Signaling, 9145). Secondary antibodies were Alexa Fluor labeled (Alexa Fluor 488 and 594) and purchased from Dianova, Hamburg, Germany. To detect acidic cell compartments, infected MF were incubated with equilibrated 1 mM LysoTracker Red DND-99 (Life Technologies) diluted in RPMI-CM one hour prior to fixation. As a phagolysosomal marker, DQ-Red BSA (Life Technologies) was used at a concentration of 2 mg/ml and incubated with the infected MF 4 hours prior to fixation.
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