Wave fx spinning disc confocal microscope

Detection of cellular proteins was performed using immunofluorescence as described previously [33 (link)]. Cells were cultured on 180-μm-thick polymer coverslip 8-well plates (μ-Slide ibiTreat plates #80826) and treated as appropriate. After 24 h, cells were fixed using 4% paraformaldehyde. Cells were permeabilized using 0.1% Triton in PBS, blocked using Odyssey blocking buffer, and incubated with primary antibody overnight at 4 °C. Primary antibody binding was detected using AlexaFluor 488 goat anti-mouse IgG (Abcam 1:500); AlexaFluor 647 goat anti-rabbit IgG (1:500); or AlexaFluor 568 goat anti-goat IgG (Abcam, 1:500). Cells were stained with DAPI and mounted using Prolong^TM Gold antifade reagent (Invitrogen, #P36934). Slides were imaged using a Wave FX spinning disc confocal microscope (Zeiss) with Volocity 6.3 acquisition and analysis software (Perkin Elmer), and basic contrast enhancement performed using unbiased automatic black-point calculation. Composite z-stack images included 15-20 XY planes over a total vertical distance of 4–6 μm using the Improvision Focus Drive. All cells were imaged using a × 40 oil immersion objective lens and quantification was performed on a single XY plane per field of view.

Slides were deparaffinized by incubation for 1 hour at 60 degrees followed by one 10 minute and 2 five minute incubations in xylene baths through decreasing concentrations of ethanol to distilled water. Antigen retrieval was performed by boiling in 10 mM sodium citrate (pH 6.0) 1hr. Slides were blocked with HHFH buffer (1 mM HEPES buffer, 2% (v/v) horse serum, 5% (v/v) FBS, 0.1% (w/v) sodium azide in Hank’s balanced salt solution (HBSS)) for 4 hours at room temperature. Slides were incubated with a cocktail of rabbit anti-GFAP or anti-Iba-1 (1:400), MAB995 against peptidoglycan (1:150) overnight at four degrees Primary antibody was removed by three 5 min PBS washes and slides were incubated for three minutes in 0.22 micron filtered 1% (w/v) Sudan black in 70% ethanol and washed an additional 3 times in PBS. A cocktail of 1:500 Alexa 488 goat anti rabbit IgG, Alexa 568 goat anti mouse IgG for two hours, washed three times in PBS stained with DAPI for 10 minutes, washed 3 times in PBS and mounted with Prolong gold antifade reagent. Slides were imaged with wave fx spinning disc confocal microscope (Zeiss). Total GFAP and Iba-1 positive cells were counted in ten fields per case at 20X and the number of cells with attached peptidoglycan immune positive particles and internalized particles were counted.