The Nebraska Transposon Mutant Library (NTML) was constructed in LAC JE228 (link). Δddh, Δldh1, Δldh2, and ΔlctP1 Tn mutants were moved to the USA300 LAC 13c background by transduction with bacteriophage φ1128 (link) (Extended Data 1a ). Lactate double (Δldh1/ldh2) and triple (Δddh/ldh1/ldh) mutants were constructed from each single Tn mutant after switching antibiotic resistance cassettes associated with the Tn mutant. Transposon insertions were confirmed by PCR using chromosomal primers flanking the gene containing the Tn insertion. All S. aureus strains were transduced with a GFP plasmid (pCM13)91 (link) and imaged using a Zeiss 710 META laser scanning microscope (Carl Zeiss) after 4 days of biofilm growth to evaluate biofilm structure.
710 meta laser scanning microscope
Manufactured by Zeiss
Sourced in Germany
The Zeiss 710 META laser scanning microscope is a high-performance imaging system designed for advanced microscopy applications. It utilizes a multipoint scanning technique and offers a range of laser wavelengths for a versatile imaging experience. The Zeiss 710 META provides users with a powerful tool for studying samples at the microscopic level.
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3 protocols using 710 meta laser scanning microscope
1
Constructing S. aureus Lactate Mutants
2
Constructing S. aureus Lactate Mutants
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The Nebraska Transposon Mutant Library (NTML) was constructed in LAC JE228 (link). Δddh, Δldh1, Δldh2, and ΔlctP1 Tn mutants were moved to the USA300 LAC 13c background by transduction with bacteriophage φ1128 (link) (Extended Data 1a ). Lactate double (Δldh1/ldh2) and triple (Δddh/ldh1/ldh) mutants were constructed from each single Tn mutant after switching antibiotic resistance cassettes associated with the Tn mutant. Transposon insertions were confirmed by PCR using chromosomal primers flanking the gene containing the Tn insertion. All S. aureus strains were transduced with a GFP plasmid (pCM13)91 (link) and imaged using a Zeiss 710 META laser scanning microscope (Carl Zeiss) after 4 days of biofilm growth to evaluate biofilm structure.
3
Immunofluorescence Analysis of Skin Abscess
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Tissues from the skin abscess or surrounding sterile field were processed for immunofluorescence staining using primary antibodies for matrix metallopeptidase 9 (monoclonal mouse anti-MMP9; Abcam) and collagen type I (polyclonal rabbit anti-ColI; Millipore, Burlington, MA). Frozen tissue sections were fixed (100% methanol), blocked in 5% donkey serum, and incubated with primary antibodies (Arg-1, 1:100; iNOS, 1:100; MMP-9, 1:500; ColI, 1:40) diluted in 2% donkey serum overnight at 4°C. Sections were then incubated with donkey anti-mouse Alexa Fluor 488 and donkey anti-rabbit Alexa Fluor 648 (1:250; Jackson ImmunoResearch Laboratories, West Grove, PA) secondary antibodies and Hoechst stain (1:100; Molecular Probes, Eugene, OR) for 2 h at room temperature. The samples were washed and dried, and the glass slides were mounted with Slow Fade (Life Technologies, Carlsbad, CA). Confocal imaging was performed using a Zeiss 710 META laser scanning microscope (Carl Zeiss, Oberkochen, Germany).
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