Bu içeriği görüntülemek için JoVE aboneliği gereklidir.  Oturum açın veya ücretsiz deneme sürümünü başlatın.
Characterization of Macrophage Extracellular Traps in Mouse Lung Tissue Samples

Characterization of Macrophage Extracellular Traps in Mouse Lung Tissue Samples

DEŞİFRE METNİ

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.

İlgili Videolar

Read Article