Immunofluorescence Labelling of Human and Murine Neutrophil Extracellular Traps in Paraffin-Embedded Tissue

Neutrophil extracellular traps (NETs) are three-dimensional structures generated by stimulated neutrophil granulocytes. It has become clear in recent years that NETs are involved in a wide variety of diseases. Detection of NETs in tissue may have diagnostic relevance, so standardized protocols for labelling NET components are required.

Today we'd like to show you variants of the common heat-induced antigen retrieval protocol. It combines the benefits of lower temperature for the neutrophil elastase antigen and high pH value that is required for histones. This technique allows to study NETs, neutrophil extracellular traps, in paraffin tissues both from mouse or men.

And it's also considered to study archived material or retrospective study. First, place the prepared slides in racks and submerse them into the media used for dehydration and clearing in reverse order for five minutes each. Next, heat a water bath with a temperature-controlled hot plate to 70 degrees Celsius.

Place a jar filled with heat-induced epitope retrieval buffer and 10%glycerol into the water bath. When the buffer has reached 70 degrees Celsius, place the rack with the slides into the buffer jar. Incubate the slides at 70 degrees Celsius for 120 minutes.

After this, remove the jar from the water bath, and let it cool to room temperature. Rinse the cooled sections three times with the ionized water and one time with TBS. Using rolled filter paper or a cotton swab, carefully remove any liquid between the sections on the slides, making sure to leave the sections hydrated.

Then, use a hydrophobic barrier pen to create a barrier around each section. Incubate the section in blocking buffer at room temperature for 30 minutes to prevent unspecific binding. Dilute the primary antibodies in blocking buffer at a concentration of one microgram per milliliter.

Remove the blocking buffer from the slides, and add the diluted primary antibodies, making sure to use a sufficient volume to prevent drying. Close the moist container, and incubate overnight at room temperature. The next day, wash the sections three times with TBS with each wash lasting five minutes.

Prepare a working solution of secondary antibodies in blocking buffer. Cover the tissues sections with the secondary antibody solution, and transfer the slides to a moist container. Seal the moist container, and incubate at room temperature for one hour.

After this, wash the sections three times with TBS and one time in water with each wash lasting five minutes. Cover the washed sections with mounting medium, and apply cover glass while avoiding bubble formation. After the mounting medium has solidified, use a confocal microscope or a wide-field microscope with appropriate band pass filters to analyze the immunofluorescence.

To digitize the complete sections using a slide scanner, set the fluorescence intensity using the negative and positive controls. Using the neutrophil elastase staining, find the neutrophil-rich areas and zoom in on those areas to check if the neutrophil elastase signal is granular or extracellular. If the signal is extracellular and overlaps with both histone and DNA staining, the neutrophil extracellular traps have been obtained.

In this study, NET components are successfully detected in paraffin-embedded tissue, both of human and murine origin. If the tissue sections have a thickness between two to three micrometers, they can be analyzed by wide-field microscopy using 10x or 20x objectives. To correctly evaluate the staining, both negative and positive samples have been processed.

A representative section of human appendicitis tissue shows tissues stained for NE, H2B, and Hoechst 33342. The images on the left are from an area of the section containing NETs, while the images on the right are from a different area of the same section that contains numerous neutrophils but no NETs. Areas with massive NET formation can easily be found even at low magnifications since all three NET components colocalize often in stringy extracellular structures.

This appears in the overlay of the three channels as whitish extracellular fibers which can be quantified by using image analysis software to create a purple overlay using pixels from overlapping signals that are positive for green, red, and blue. For higher resolution, confocal microscopes or wide-field microscopes with deconvolution have to used to minimize out-of-focus blur. A maximum projection of a confocal stack of a NET-rich area from the same human appendicitis specimen shows that NE is found in granules but is also abundant extracellularly where it colocalizes with H2B and with DNA.

The extracellular colocalization results in a whitish color combination. The pixels positive for green, red, and blue can once again be used to create a purple overlay presenting NETs. A representative detail of a central section from a mouse lung infected with Mycobacterium tuberculosis provides another example of the colocalization of all three NET components being clearly visible as whitish areas between neutrophils which can be used to create a purple layer indicating NETs.

During the staining procedure, the sections must not fall dry because this will cause a false-positive staining or a high background. The analysis of archived material may help to understand the impact of NETs in diseases. Because paraffin tissue can have a huge background, it is important to have a positive and a negative control that you can distinguish between your staining and the background.

Dewaxing and dehydration should be performed under a fume hood. Make sure to wear gloves and your lab coat.