Characterization of Macrophage Extracellular Traps in Mouse Lung Tissue Samples
Transcript
To pre-treat the FFPE samples with antigen retrieval solution, oven-dry the slides at 60 degrees Celsius for 60 minutes. Then, transfer the slides into xylene solution for 30 minutes, before transferring the samples to 70% ethanol at room temperature for 5 minutes.
After using tap water to rinse the slides, place them in heat-proof plastic wrap, and subject them to higher antigen retrieval by placing them in a pressure cooker in Tris-EDTA pH 9.0 for 10 minutes. Cool the samples for 20 minutes, and transfer them to tap water. Then, place them on a rocker for 5 minutes. Repeat the tap water wash, and then, wash the slides once in PBS on the rocker.
To block the samples, add 10% chicken serum in 5% BSA/PBS, and incubate them at room temperature for 30 minutes. Then, to define macrophage extracellular traps, or METs in macrophages, add a 1-to-100 dilution of primary antibodies in 1% BSA/PBS, and incubate the slides at 4 degrees Celsius for 16 hours.
Use PBS to wash the samples two times on a rocker for 5 minutes each. Add the corresponding fluorescent secondary antibodies in 1% BSA/PBS, and incubate the slides at room temperature for 40 minutes. Following PBS washes, use DAPI-containing mounting medium to stain the chromatin and mount the samples.
Using a confocal laser-scanning head attached to an inverted microscope, capture fluorescent images using 20X 0.1 NA air and 40X 1.0 NA oil objectives. Capture single-plane 512-by-512 pixel images by clicking on the line-sequential, leveling button. Obtain at least 10 fields of view per section for analysis and data for each result.
