Schematic of cell structure with expanded view of a peroxisome

Peroxisome structures are single membrane-bound vesicles that are involved in energy metabolism and lipid biosynthesis. Here we describe peroxisome stains using organelle-specific fluorescent fusion proteins in live cells or antibodies to peroxisome membrane proteins in fixed cells.

See peroxisome stains selection guide

Peroxisomes introduction

Peroxisomes are single membrane–bound vesicles found in most eukaryotic cells. They contain several different enzymes and are involved in various metabolic pathways [1]. Most importantly, peroxisomes are involved in the oxidation of fatty acids, a major source of metabolic energy. Additionally, peroxisomes catalyze the breakdown of reactive oxygen species (ROS), specifically hydrogen peroxide (H2O2), by converting it to water, or using it to oxidize other organic compounds [1,2]. Peroxisomes are also critical in the biosynthesis of phospholipids involved in maintaining the normal function of both brain and lungs in mammals.

Selection guide for peroxisome stains

 
Readout
 Expression of fluorescent fusion protein
 Secondary staining of peroxisomes
Target
Uses peroxisomal C-terminal targeting sequence  
Labels peroxisomal membrane protein 70 (PMP70)
Common filter set
FITC
FITC
Labels
GFP
Alexa Fluor 488
Ex/Em (nm)
488/520
496/519
Signal-to-noise ratio
Photostability
Multiplexing
Yes
Yes
Live cells
Yes
No
Fixed cells
No
Yes
Fixable
Yes
No
Platform
 Imaging
Imaging
Format
1 mL
1 mL
Cat. No.

Live cell peroxisome label

CellLight fluorescent fusion proteins can be used as peroxisome labels in live cells and to follow the dynamics of intracellular behavior. CellLight Peroxisome proteins are ready-to-use constructs that use the human peroxisomal C-terminal targeting sequence (0.3 kDa) to target the peroxisome.

Introducing CellLight fluorescent proteins involves a simple transfection step using the BacMam technology, and they work like cell stains with minimal toxicity or chemical disruption. These peroxisome fusion proteins are compatible with other fluorescent probes for multiplex analysis in live cells (Figures 1 and 2), or after formaldehyde fixation for colocalization studies.

Microscopic image of a cell stained with green peroxisomes, red mitochondria, and blue nucleus
Figure 1. Multiplex with CellLight Peroxisome-GFP. HeLa cells were transduced with CellLight Peroxisome-GFP. The following day, nuclei were stained with Hoechst 33342 and mitochondria were stained with MitoTracker Red CMXRos. Live-cell imaging was performed using a 63X water-immersion objective.
Microscopic image of cells stained with green peroxisomes then fixed and stained for red prohibition gene and blue nuclei
Figure 2. Multiplexing with CellLight Peroxisome-GFP using Alexa Fluor 594 Tyramide SuperBoost Kit. HeLa cells were treated with CellLight Peroxisome-GFP, BacMam 2.0 to express GFP in peroxisomes. Cells were fixed and permeabilized with Image-iT Fixation/Permeabilization Kit. Cells were incubated with Anti-Prohibiton antibody and labeled with Alexa Fluor 594 Tyramide SuperBoost Kit, Goat anti-Mouse IgG. Nucleus was labeled with NucBlue Fixed Cell ReadyProbes Reagent. Images were taken on a confocal microscope.

Fixed cell peroxisome label

The SelectFX Alexa Fluor 488 Peroxisome Labeling Kit provides the reagents needed to label peroxisomes in fixed cells, including cell fixative and permeabilization reagents (Figure 3). For detection, the kit employs a primary antibody directed against peroxisomal membrane protein 70 (PMP70), which is a high-abundance, integral-membrane component of peroxisomes. PMP70 is significantly induced by administration of hypolipidemic agents in parallel with peroxisome proliferation and the induction of peroxisomal fatty acid beta-oxidation enzymes.

Microscopic image of cells multiplexed with green peroxisome antibody, red mitochondria reagent, and blue nuclear stain
Figure 3. Bovine pulmonary artery endothelial cell labeled with probes to visualize mitochondria, peroxisomes, and the nucleus. Mitochondria were stained with the MitoTracker Red CMXRos reagent. Peroxisomal labeling was achieved with the SelectFX Alexa Fluor 488 Peroxisome labeling kit which contains a primary antibody directed against PMP70, visualized using green-fluorescent Alexa Fluor 488 goat anti-rabbit IgG. The nucleus was stained with blue fluorescent DAPI.

Learn more about peroxisome stains

References

  1. Cooper GM (2000) The Cell: A Molecular Approach. 2nd edition. Sunderland (MA): Sinauer Associates. Peroxisomes.
  2. Islinger M and Schrader M (2011). Curr Biol. 21(19):R800-1. PMID: 21996497.

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