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

Summary

Several different methods have been established for multiplex immunostaining using primary antibodies from the same host species. Here, we describe the use of microwave-mediated antibody stripping and of fluorophore-tyramide to block antibody cross-reactivity during multiplex immunostaining on formalin-fixed paraffin-embedded mouse adrenal sections.

Abstract

Immunostaining is widely used in biomedical research to show the cellular expression pattern of a given protein. Multiplex immunostaining allows labeling using multiple primary antibodies. To minimize antibody cross-reactivity, multiplex immunostaining using indirect staining requires unlabeled primary antibodies from different host species. However, the appropriate combination of different species antibodies is not always available. Here, we describe a method of using unlabeled primary antibodies from the same host species (e.g., in this case both antibodies are from rabbit) for multiplex immunofluorescence on formalin-fixed paraffin-embedded (FFPE) mouse adrenal sections. This method uses the same procedure and reagents used in the antigen retrieval step to strip the activity of the previously stained primary antibody complex. Slides were stained with the first primary antibody using a general immunostaining protocol followed by a binding step with a biotinylated secondary antibody. Then, an avidin-biotin-peroxidase signal development method was used with fluorophore-tyramide as the substrate. The immunoactivity of the first primary antibody complex was stripped through immersion in a microwaved boiling sodium citrate solution for 8 min. The insoluble fluorophore-tyramide deposition remained on the sample, which allowed the slide to be stained with other primary antibodies. Although this method eliminates most false positive signals, some background from antibody cross-reactivity may remain. If the samples are enriched with endogenous biotin, a peroxidase-conjugated secondary antibody may be used to replace the biotinylated secondary antibody to avoid the false positive from recovered endogenous biotin.

Introduction

In multiplex immunostaining, direct staining using conjugated primary antibodies can provide informative results. Without using secondary antibodies, the direct staining method has a low risk of false colocalization signals from antibody cross-reactivity. However, the conjugated reporters (fluorophore, enzymes) or biotin on the primary antibody limit its future use. Alternatively, indirect immunostaining usually provides stronger signals by using an unconjugated primary antibody with a labeled secondary antibody. Ideally, unconjugated primary antibodies used in multiplex immunostaining should come from different host species to avoid antibody cross-reactivity. However....

Protocol

1. Staining with the First Antibody

1. Dewax and rehydrate FFPE slides with 5 min allotted to each of the following steps: xylene or equivalent reagents 3x, 100% ethanol 2x, 95% ethanol 1x, 70% ethanol 1x, 50% ethanol 1x, and distilled water 2x.
   NOTE: Slides should remain moist starting from this rehydration step until mounting in the final step.
2. For optional antigen retrieval, place the slides in 275 mL of boiling sodium citrate solution (10 mM, pH = 6.0) for 8 min. To keep the solution boiling.......

Discussion

Multiplex immunostaining is useful to examine the cellular colocalization of two or more antigens. This widely used technique gives convincing colocalization results when primary antibodies are conjugated with different reporters (direct staining). However, direct staining usually provides weaker signals compared to indirect staining, which involves conjugated secondary antibodies to detect the primary antibodies. In indirect staining, a high-quality multiplex immunostaining result relies on whether the secondary antibod.......

Materials

Name	Company	Catalog Number	Comments
Antifade Mounting Medium	Vector Laboratories	H-1000
Biotinylated donkey anti-mouse	JacksonImmuno	715-066-151	1:500 dilution
Biotinylated donkey anti-rabbit	JacksonImmuno	711-066-152	1:500 dilution
DAPI	BioLegend	422801	2 μg/mL in distilled water
Fluorescence microscope	ECHO	Revolve 4
Horseradish peroxidase-conjugated streptavidin	JacksonImmuno	016-030-084	1:1000 dilution
Microwave oven, 700W	General Electric	JEM3072DH1BB
Mouse anti-CYP2F2	Santa Cruz,	SC-374540	1:250 dilution
Mouse anti-TH	Santa Cruz,	SC-25269	1:1000 dilution
Normal donkey serum	JacksonImmuno	017-000-121	2% serum in PBST
Rabbit anti-20αHSD	Kerafast,	EB4002	1:500 dilution
Rabbit anti-3βHSD	TransGenic,	KO607	1:250 dilution
Rabbit anti-TH	NOVUS,	NB300-109	1:1000 dilution
Rabbit anti-β-catenin	Abcam,	ab32572	1:500 dilution
Streptavidin Horseradish Peroxidase (SA-HRP)	JacksonImmuno	016-303-084	1:1000 dilution
TSA Cy3 Tyramide	PerkinElmer	SAT704B001EA	1:100 dilution
TSA Fluorescein Tyramide	PerkinElmer	SAT701001EA	1:100 dilution