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

Published: April 30, 2024

Abstract

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

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines. 1. Staining with the First Antibody 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 th…

Divulgations

The authors have nothing to disclose.

Materials

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

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Citer Cet Article
A Multiplex Immunostaining Method Using Primary Antibodies from the Same Host Species. J. Vis. Exp. (Pending Publication), e22138, doi: (2024).

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