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