Identification of Neutrophil Extracellular Traps in Paraffin-Embedded Feline Arterial Thrombi using Immunofluorescence Microscopy

This protocol of the parafinization followed by antigen retrieval of paraffin-embedded aortic sections can be a useful tool to study the role of neutrophilic tricellular traps in feline thrombosis. Detection of neutrophilic tricellular traps by immunofluorescence in fixed and paraffin-embedded tissue, is superior to conventional histological stents like HNE, as it allows simultaneous detection of safer DNA and extracellular proteins. This protocol can be used in other type of thrombi and in other veterinary species.

Identification of neutrophil extracellular traps within feline arterial thrombi suggest that they may play a role in thrombosis in cats. Protocols presented here are guidelines. We strongly encourage investigators to adjust the duration and conditions of the antigen retrieval process to yield a satisfactory signal.

To use an automated system to perform deparaffinization and re-hydration of the sections on glass slides, place the glass slides in racks. Submerge completely in 100 percent saline for three minutes. Repeat this step twice.

Do not rinse in between steps. Submerge completely in decreasing concentrations of ethanol at room temperature three times for three minutes each. Submerge completely in deionized water for two minutes and repeat one more time.

After treatment with TBST, fill the reservoir with deionized water heated to 100 degrees Celsius. Allow the steamer chamber to equilibrate for 20 minutes. On a heat plate, heat the commercially available antigen retrieval solution containing Tris and EDTA at pH 9.0 to 95 to 97 degrees Celsius.

Ensure that it does not boil. Suberge the slides completely in the heated antigen retrieval solution and continue the application of external heating via the steamer for 20 minutes. Next, wash the slides three times with TBST for five minutes.

Now, place the sections in blocking buffer 1. Incubate for two hours at room temperature under gentle rocking between 30 and 50 rpm. Without washing, immediately apply 100 microliters of diluted rabbit polyclonal anti-human citrullinated histone H3 antibody directly onto the slide.

Place a cover slip on each section to allow even distribution of the antibody mixture. Incubate for 12 to 16 hours at four degrees Celsius with gentle rocking. After that, wash the slides three times with TBST for five minutes each time.

To apply antibody, use a pipet and evenly distribute 100 microliters of goat anti-rabbit antibody conjugated to Alexa Fluor 488. Cover the slides with tin foil and incubate the slides for one hour at room temperature under gentle rocking. After washing with TBST according to the manuscript, incubate the sections in blocking buffer 2 overnight at four degrees celsius under gentle rocking.

Protect from the light. Wash with TBST three times for five minutes each time. Block the sections in blocking buffer 3 as done previously at room temperature for two hours under gentle rocking.

Incubate the sections with biotin laded ployclonal rabbit anti-human neutrophil elastase antibody at four degree Celsius for 12 to 16 hours as described previously. After washing with TBST incubate the slides with Alexa Fluor 594 Streptavidin Conjugate for one hour at room temperature. After that, wash with TBST one time for five minutes.

Apply 100 microliters of Autofluorescense Quenching solution mixture directly onto the sections for one minute as instructed by the manufacturer. Immediately wash the slides with TBST six times for 10 minutes each time. Cover each slide with 100 microliters of 300 nanomolar DAPI for five minutes in the dark.

Then wash with TBST five times for three minutes each time. Apply a drop of antifade mounting medium directly onto the glass slide surrounding the section. Place a cover slip gently onto the section without creating any bubbles.

Allow the samples to cure overnight in the dark at four degrees Celsius. To locate thrombi, scan cranially to caudally along the length of the aorta, aortic bifurcation and each femeral artery using phase contrast microscopy with a 10x objective. First examine the sections for cell-free DNA using the DAPI channel with the excitation at 357 and 44 nanometers.

Identify extracellular neutrophil elastase and citrullinated histone H3 on the Texas red and green fluorescent protein channels respectively. Maintain consistent exposure time and gains of each channel throughout the acquisition of images to avoid saturation in pixel intensity. Neutrophil extracellular traps are identified based co-localization of neutrophil elastase, citrullinated histone H3 and cell-free DNA.

Using hematoxylin and eosin stain, and brightfield microscopy, feline arterial thrombi consist of red blood cells, leukocytes, fibrin and platelets. NETs appeared as networks of deep purple threads of various lengths surrounding nearby erythrocytes and leukocytes. Using this protocol, immunofluorescence microscopy revealed large aggregates of NETs composed of cell-free DNA, extracellular citrullinated histone H3 and neutrophil elastase.

Under phase contrast in immune microscopy, a thrombus was characterized as a well-demarcated structure within the vascular space. However, thrombi were not detected in any of the control samples. This figure demonstrates profound autofluoresence of clot elements like erythrocytes when imaged at the 488 nanometer wavelength.

Brief autofluoresence quenching after immunolabelling significantly increased the sensitivity of protein co-localization and NET detection even in areas with an abundance of erythrocytes. The duration of fixation effects the immunoreactivity of certain antigens. We recommend fixation for no longer than 24 hours par the dehydration and paraffin embedding.

Alternatively, methanol-free paraformaldehyde can be used to limit artifact by formic acid and ketones from oxidation of formaldehyde. To prevent interference when using primary antibodies originating from the same species, we included additional blocking step to saturate any remaining binding sites on the secondary antibodies. Investigators must include negative controls consisting of isotype antibodies, exclusion of either primary antibodies and immuno-labeling step and biological controls from cats without thrombosis.

Autofluoresence of tissue limits of thrombi can hammer the microscopic identification of neutrophil extracellular traps. Investigator can minimize the fluorescence by the use of far-red fluorophores. This technique can be a valuable tool for the study of neutrophil extracellular traps in other veterinary species.

This can provide a better understanding of the pathophysiology of thrombosis.