A Multiplex Immunostaining Method Using Primary Antibodies from the Same Host Species

Take a fixed and permeabilized adrenal section comprising a cortex and a medulla. Treat it with a boiling acidic buffer, breaking the fixation-induced crosslinks for antigen unmasking.

Block the non-specific binding sites and introduce primary antibodies binding to cortex-specific markers.

Add biotinylated secondary antibodies targeting the primary antibodies and streptavidin-conjugated peroxidases binding to biotin.

Introduce a fluorophore-tagged tyramide and hydrogen peroxide.

The immobilized peroxidases utilize hydrogen peroxide to activate tyramide, which binds to nearby proteins, labeling the cortex.

Next, treat the section with the boiling buffer to strip the bound antibodies.

Introduce the same host species-generated primary antibodies targeting medulla-specific markers and biotinylated secondary antibodies.

Introduce streptavidin-conjugated peroxidases and a different fluorophore-conjugated tyramide to label the medulla.

Stripping the cortex-specific primary antibodies inhibits the reintroduced secondary antibody binding them, preventing cross-reactivity.

Apply a nuclear stain and perform imaging to visualize the distinctly stained cortex and medulla, confirming no antibody cross-reactivity.

Before staining, dewax and rehydrate formalin-fixed, paraffin-embedded slides with five-minute immersions in each solution as indicated. After the second distilled water immersion, place the slides in 275 milliliters of boiling sodium-citrate solution on the bottom of a pipette tip box with a lid, and place the box into a 700-watt microwave at 70% power for eight minutes.

At the end of the treatment, open the lid and allow the solution to cool to room temperature before washing the slides with three five-minute washes of fresh PBST in a Coplin jar. To block any nonspecific binding sites, after the last wash, shake the excess PBST from each slide and cover the slides with a sufficient volume of an appropriate blocking solution.

After 30 minutes at room temperature in a humidified chamber, shake the excess blocking solution from each slide, and add 250 microliters of the first primary antibody of interest to each sample. To prevent samples from drying out, the slides may be covered by a piece of paraffin film.

After an overnight incubation at four degrees Celsius in the humidified chamber, wash the slides three times with fresh PBST for five minutes per wash. Shake off the excess PBST, then, quickly cover each slide with 250 microliters of an appropriate secondary antibody solution for a one-hour incubation in the humidified chamber at room temperature.

At the end of the incubation, wash the slides three times in fresh PBST as demonstrated, and add 250 microliters of freshly prepared streptavidin horseradish peroxidase to each slide. After 30 minutes at room temperature, wash the slides three times in fresh PBST, then, shake off the excess PBST, and cover each slide with 250 microliters of freshly prepared fluorophore tyramide solution.

One minute later, submerge the slides in fresh PBST, followed by three two-minute washes in fresh PBST. After the last wash, apply a few drops of a 1-to-1 glycerol to PBS solution to the sections, and check for the presence of a fluorescent signal by fluorescence microscopy.

To strip the first antibody complex, place the antibody-labeled slides in 275 milliliters of boiling sodium-citrate solution for at least eight minutes as demonstrated. At the end of the treatment, open the lid and let the solution cool to room temperature, before washing the slides three times with PBST for five minutes per wash.

To stain for the second target protein of interest, after blocking for nonspecific binding is demonstrated, label each sample with 250 microliters of the second primary antibody of interest at four degrees Celsius overnight. The next morning, wash the slides three times for five minutes in fresh PBST per wash before covering each slide with 250 microliters of an appropriate secondary antibody solution for a one-hour incubation at room temperature.

At the end of the incubation, wash the slides three times in fresh PBST as demonstrated, and add 250 microliters of freshly prepared streptavidin horseradish peroxidase to each slide. To develop the signal for the second target protein of interest after three PBST washes, treat the slides with a fluorophore-conjugated tyramide in a different fluorescent spectrum.

To stop the tyramide signal development, submerge the slides into PBST then stain the samples with an appropriate nuclear dye.