Microwaving and Fluorophore-Tyramide for Multiplex Immunostaining on Mouse Adrenals − Using Unconjugated Primary Antibodies from the Same Host Species

Immunostaining is widely used in biomedical research to identify the location of a protein of interest. Multiplex immunostaining can detect multiple targets on the same sample using different primary antibodies. The benefit of this method is the use of commonly available buffers to eliminate antibody cross-reactivity allowing immunostaining using two or more unlabeled primary antibodies from the same host species.

Demonstrating the procedure will be Sophia Zheng, a graduate student, and Qiongxia Lyu, a visiting scholar of my lab. Before staining, de-wax 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 75%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 a 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 a 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 one-to-one glycerol-to-PBS solution to the sections and check for the presence of a fluorescence 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 as 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 the fluorophore conjugated tyramide in a different fluorescence spectrum. To stop the tyramide signal development, submerge the slides into PBST.

Then stain the samples with an appropriate nuclear dye. For imaging by fluorescence microscopy, mount each nuclear dye labeled slide with a drop of an appropriate mounting medium in a coverslip. Use the 4X objective to locate the tissue on the slide.

Switch to the 10X objective for imaging and adjust the exposure time to 100 to 400 milliseconds with the light source between 40 to 80%intensity. Then obtain images in each channel without moving the stage. The images from each channel can be merged to form a multiplex fluorescent image.

Without stripping between the first and secondary antibody staining, the second staining for tyrosine hydroxylase will pick up signals from the first target protein of interest resulting in a false positive tyrosine hydroxylase staining, for example in this sample within the adrenal cortex. To remove the antibody cross-reactivity in the horseradish peroxidase from the first immunostaining, one-minute and eight-minute microwave-mediated stripping can be performed resulting in clean double staining results. No significant signal is picked up in negative control samples.

Eight-minute stripping in boiling citrate buffer is also sufficient to eliminate antibody cross-reactivity for many antibodies commonly used in the adrenal gland. In some cases, a weak false positive signal is still detectable, however, and an increase of 20 minutes to the microwave treatment still may not completely remove the antibody cross-reactivity. During fluorescence imaging, it is important not to move the stage when switching channels.

However, it may be necessary to readjust the focus to obtain clear images of each channel. For each channel, be sure to adjust the exposure time to ensure that the signals are appropriately exposed with no overexposed pixels or areas in the images. During the staining procedure, it is important to keep the slides hydrated from the de-waxing and The slides can be stripped using the same stripping methods several times to allow multiplex staining.