Direct Detection of Isolevuglandins in Tissues Using a D11 scFv-Alkaline Phosphatase Fusion Protein and Immunofluorescence

Isolevuglandins have been implicated in several diseases, particularly cardiovascular disease. This protocol was developed to detect isolevuglandins in tissues with D11 alkaline phosphatase fusion protein, and immunofluorescence. Current methods of detection are expensive or require a secondary antibody for an E-tag.

Finding a reliable secondary antibody is difficult, but this protocol removes the secondary antibody step. immerse the slides of mouse and human paraffin-embedded tissues in xylene three times for five minutes to deparrafinize the tissues. for rehydrating the tissue, wash the tissues two times with 95%ethanol, followed by two washes with 70%ethanol and then twice with 50%ethanol prepared in water.

Wash the slides in tris-buffered saline with 0.1%Tween 20 three times by filling the slide holder with TBST. Then discard the TBST. Place the slides in preheated sodium citrate buffer and incubate in a pressure cooker set to four minutes on high pressure, for a total antigen retrieval time of 20 minutes.

At the end of the incubation, remove slides from the pressure cooker and allow them to cool for 20 minutes at room temperature. Give three quick washes to slides with TBST as demonstrated. Block the slides by adding 2%BSA dissolved in TBST.

Cover the slides with paraffin tape and incubate at room temperature for 15 minutes. After incubation, discard the blocking buffer. Add 200 microliters of D11 alkaline phosphatase prepared in TBST to the slides and cover the slides with paraffin tape.

After incubating the slides in a humidified chamber for three hours at room temperature, wash the slides three times with TBST. Develop the slides with a calorimetric alkaline phosphatase developer for immunohistochemistry, or a fluorescent alkaline phosphatase developer for immunofluorescence. Wash slides once with TBST to remove the excess developer and prevent further color development.

Counterstain the slides with one microgram per milliliter HEX nuclear stain prepared in PBS for immunofluorescence. Then wash the slides once with TBST to remove any excess counterstain. Use the mounting medium to place the cover slip on the developed slides.

Observe the slides under an inverted light microscope for immunohistochemistry, or a confocal fluorescent microscope for immunofluorescence. Four negative control experiments can be performed to confirm the specificity of D11 AP staining for isolevuglandin. In the first negative control experiment, incubate the tissues with D11 AP diluted in TBST, or TBST alone.

Then incubate the tissues with diluted D11 AP and bacterial pariplasmic extract diluted in TBST without the D11 AP linker. For the competitive assay, prepare isoLG and isoLG adducted to mouse serum albumin at a molar ratio of eight to one. Dilute D11 AP one to 10 in TBST.

Then incubate the diluted D11 AP with 50 micrograms per milliliter isolevuglandin-adducted MSA, or non-adducted MSA for one hour at room temperature. Add D11 AP with isolevuglandin MSA or D11 AP with non-adducted MSA to tissues for staining. Use a relevant single chain fragment variable antibody D20 to stain the tissues for the final negative control set.

Immunofluorescence of the aorta in hypertensive and normotensive mice was studied using this protocol. Staining with D11 AP indicated an increased concentration of isoLGs in the aorta of mice with angiotensin II-induced hypertension compared to the control mice. The D11 AP was used to detect the isolevuglandins present in the intestinal tissues of human patients with hypertension and normotensive humans.

The tissues from the patients with hypertension had elevated concentrations of isolevuglandins. Tissues were stained with pariplasmic extract and without D11 AP.The brighter staining was observed in the tissue stained with D11 AP compared to the pariplasmic extract-stained tissue, confirming that the staining was not due to the non-conjugated bacterial alkaline phosphatase present in the pariplasmic extract. In the competitive assay, the tissue stained with D11 AP pre-incubated with the isoLG competitor showed diminished staining, similar to the staining observed in tissues with D11 AP, which shows specificity to D11 AP to isolevuglandins.

In the negative control, staining the mouse aorta with D11 AP resulted in strong staining compared to D20, indicating the specificity of D11 AP to isolevuglandins in the hypertensive aorta. It's important to inactivate any endogenous alkaline phosphatase present in tissue to limit background staining.