Peroxisome Staining in Mammalian Cells Using Peroxisome-Specific Probes

So our research focuses on understanding peroxisomes, which are essential organelles for lipid metabolism and cellular oxidative balance. We aim to uncover how their structure and function are regulated and how these processes change in disease. To achieve this, we develop fluorescent probes that allow us and the wider scientific community to visualize peroxisomes in a range of animal cells.

Here, we develop staining protocols that work in both live and fixed cells, providing powerful tools for studying paroxetine biology in health and pathology. One of the main experimental challenges is achieving non-invasive labeling of peroxisomes in animal cells. Whilst antibody staining helps overcome this, there is a lack of commercially available antibodies for paroxysmal proteins.

Another challenge is visualizing peroxisomes non-invasively in real time, which requires adaptable protocols. And finally, conventional staining doesn't provide a functional readout. While peroxidase-based staining can vary depending on paroxysmal function, which is both a challenge and an opportunity, because it allows us to distinguish paroxysmal functional states within the cell.

Begin with mammalian cells seeded the day before in a plate with cover slips that are now at a 30 to 70%confluency. Dilute the peroxisome probe with fresh media in a light-sensitive, micro-centrifuged tube to achieve a four micromolar final concentration. Select the desired well to stain and label the outside using a marker pen.

Aspirate 0.5 milliliters of media from the selected well and replace it with the prepared probe media drop by drop along the wall of the well. After dispensing, incubate the cells at 37 degrees Celsius for 10 to 15 minutes. From this point forward, shield the cells from light using aluminum foil.

Post-incubation, carefully remove the stained media and wash the cells twice with PBS. Then, place the plate under a chemical hood, add 4%paraformaldehyde in PBS to the cells, and incubate at room temperature for 20 minutes. After fixation, remove the paraformaldehyde into a designated waste container and wash the cells once with PBS.

Next, add 0.5%Triton X in PBS to permeabilize the cells and incubate for 2.5 minutes. After incubation, remove the Triton X and wash the cells twice with PBS for 10 minutes each. To block the cells, incubate them with 5%bovine serum albumin in PBS overnight at four degrees Celsius.

Perform the immunofluorescent staining procedure following standard protocol. Finally, perform microscopy to acquire images of both fixed and live cells. Live cell staining with Peroxi_SPY probes resulted in ATP-binding cassette subfamily D-dependent import into peroxisomes, producing a high peroxisome to cytoplasm fluorescence ratio in metabolically-active cells.

In wild type HEK-293T cells expressing GFP-SKL, Peroxi_SPY555 staining showed distinct colocalization with peroxisomes, whereas in PEX19 knockout cells that lack peroxisomes, the staining was absent. In human fibroblasts, Peroxi_SPY staining colocalized with the paroxysmal marker GFP-SKL, confirming peroxisome targeting. Peroxisome density quantification revealed significant variation among different cell types.

Optimized staining conditions in COS-7 cells using 250 nanomolar Peroxi_SPY for 20 minutes significantly increased the peroxisome to cytoplasm fluorescence ratio compared to the original one micromolar for 10 minutes protocol. Extended incubation of HEK-293T cells with Peroxi_SPY for 24 hours led to an improved peroxisome to cytoplasm fluorescence intensity ratio compared to 15 minutes, despite reduced peroxisome staining. A fixation protocol enabled successful antibody costaining demonstrated by preserved Peroxi_SPY labeling and Anti-PEX14 immunostaining in HuH-7 and HEK-293T cells.

Quantification showed that fixation and permeabilization procedures affected peroxisome-fluorescence intensity when compared to live cell staining. We have developed the Peroxi_SPY dye that can be used to visualize peroxisomes in both live and fixed animal cells. Our fluorescent probe can offer a simple and quick method to study peroxisome dynamics, heterogeneity, and dysfunction in ways that were previously limited.

This protocol will enable imaging-based peroxisome research in diverse cell types, including patient-derived cells with paroxysmal disorders.