16S rRNA FISH was performed as previously described (Canny et al., 2006 (link)) with minor adjustments. Briefly, colon tissue sections were dissected and immediately placed in ice-cold Carnoy’s solution (Ethanol:Chloroform:Glacial acetic acid at 6:3:1) for 1–2 h. The tissue was then washed twice in 100% ethanol for 15 min followed by two washes in xylenes for 15 min then embedded in paraffin and cut to 5 mm. Slides were allowed to sit in sunlight for at least 24 h to reduce background autofluorescence. Following this, slides were deparaffinized by heating to 50°C for 1 h followed by 5 min incubations with xylene (2x) then 100% ethanol (2x) at room temperature. Slides were next incubated with a universal bacterial 16S-DNA probe (EUB-338: [Cy3]-5’-GCTGCCTCCCGTAGGAGT-3’-[Cy5]) at 0.5ng/uL in hybridization buffer (0.9M NaCl, 0.02M Tris pH 7.5, 20% Formamide, 0.05% SDS) for 2 h at 46°C. Following this incubation slides were washed in post-hybridization buffer (0.215M NaCl, 0.02 Tris pH 7.5, 0.025M EDTA, 0.05% SDS) 2x for 5 min at 46°C then rinsed with deionized water and allowed to air dry before coverslips were mounted using Antifade Mounting media with DAPI (ThermoFisher Scientific).
Antifade mounting media with dapi
Manufactured by Thermo Fisher Scientific
Antifade Mounting media with DAPI is a specialized laboratory reagent designed to preserve and protect fluorescently labeled samples. It contains an antifade agent that helps prevent photobleaching, as well as the nuclear counterstain DAPI, which binds to DNA and emits blue fluorescence.
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Lab products found in correlation
Permeabilization buffer, by BD (1 mentions)
Rabbit anti flag antibody, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 680, by Thermo Fisher Scientific (1 mentions)
Lsm 700 confocal laser scanning microscope, by Zeiss (1 mentions)
Paraformaldehyde (pfa), by BD (1 mentions)
Streptavidin pe, by Thermo Fisher Scientific (1 mentions)
2 protocols using antifade mounting media with dapi
1
Fluorescent In Situ Hybridization for 16S rRNA
2
SARS-CoV-2 Spike Protein Binding to ACE2
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T24 cells were transiently transfected with ACE2-GFP, dACE2-Myc-DDK, or co-transfected with both constructs in 4-well chambered slides (2×104 cells/well, LabTek). After 24 hrs, cells were treated with 2 ng/ml of recombinant biotinylated SARS-CoV-2 spike protein RBD (spike protein-RBD, Sino Biological) for 1 hour at 37°C. Cells were washed twice with media and then stained with 5μg/ml streptavidin PE (Thermo Fisher) for 30 min at 37°C. Cells were then washed twice with PBS and fixed with 4% paraformaldehyde (BD Biosciences) for 30 min. After rinsing twice in PBS and permeabilization buffer (BD Biosciences), cells were incubated with permeabilization buffer for 1hr. Fixed cells were incubated with rabbit anti-FLAG antibody (1:250 dilution, Thermo Fisher) overnight, washed and then stained with anti-rabbit Alexa Fluor 680 (1:500 dilution, Thermo Fisher). Slides were mounted with antifade mounting media with DAPI (Thermo Fisher) and imaged at 40X magnification on an LSM700 confocal laser scanning microscope (Carl Zeiss) using an inverted oil lens.
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