Method Article

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

July 8th, 2025

In This Article

Abstract

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Source: Lyu, Q., et al. Microwaving and Fluorophore-Tyramide for Multiplex Immunostaining on Mouse Adrenals − Using Unconjugated Primary Antibodies from the Same Host Species. J. Vis. Exp. (2020).

This video demonstrates an assay to perform multiplex immunostaining using primary antibodies from the same host species. In this method, an adrenal tissue section undergoes a two-step immunostaining process. Initially, the section is stained with antibodies specific for markers in the cortex, followed by stripping the bound antibody and restaining with antibodies specific for markers in the medulla.

Protocol

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All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Staining with the First Antibody

  1. Dewax and rehydrate formalin-fixed paraffin-embedded (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 solut....

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Disclosures

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No conflicts of interest declared.

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Antifade Mounting MediumVector LaboratoriesH-1000
Biotinylated donkey anti-mouseJacksonImmuno715-066-1511:500 dilution
Biotinylated donkey anti-rabbitJacksonImmuno711-066-1521:500 dilution
DAPIBioLegend4228012 μg/mL in distilled water
Fluorescence microscopeECHORevolve 4
Horseradish peroxidase-conjugated streptavidinJacksonImmuno016-030-0841:1000 dilution
Microwave oven, 700WGeneral ElectricJEM3072DH1BB
Mouse anti-CYP2F2Santa Cruz,SC-3745401:250 dilution
Mouse anti-THSanta Cruz,SC-252691:1000 dilution
Normal donkey serumJacksonImmuno017-000-1212% serum in PBST
Rabbit anti-20αHSDKerafast,EB40021:500 dilution
Rabbit anti-3βHSDTransGenic,KO6071:250 dilution
Rabbit anti-THNOVUS,NB300-1091:1000 dilution
Rabbit anti-β-cateninAbcam,ab325721:500 dilution
Streptavidin Horseradish Peroxidase (SA-HRP)JacksonImmuno016-303-0841:1000 dilution
TSA Cy3 TyramidePerkinElmerSAT704B001EA1:100 dilution
TSA Fluorescein TyramidePerkinElmerSAT701001EA1:100 dilution

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Tags

Multiplex ImmunostainingPrimary AntibodiesFluorophore TyramideAntibody StrippingAdrenal TissueCortex MedullaMicrowave Antigen RetrievalStreptavidin PeroxidaseNuclear StainingFluorescence Microscopy

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