Pyronin Y (Pyronine G) (Synonyms: C.I. 45005)

This plan only provides a guide, please modify it to meet your specific needs.
1. Prepare Pyronin Y staining solution
(1) Prepare Pyronin Y dye stock solution: Use DMSO/sterile water to dissolve the solid powder and prepare a 1-5mM Pyronin Y dye stock solution.
Note: The stock solution prepared with sterile water needs to be filtered with a 0.22μm filter membrane. It is recommended to aliquot Pyronin Y stock solution and store it in the dark at -20°C to avoid repeated freezing and thawing.
(2) Working solution preparation: Dilute the stock solution with a suitable buffer solution (such as HBSS or PBS) and prepare a concentration of 1-5μM Pyronin Y working fluid.
Note: Please adjust the concentration of Pyronin Y working fluid according to the actual situation and prepare it for use.
2. Cell staining
2.1 Suspension cells (taking 6-well plate as an example)
(1) Centrifuge suspended cells at 1000g for 3-5 minutes. Discard the supernatant and wash twice with PBS for 5 minutes each time.
(2) Add 1 mL of Pyronin Y dye working solution and incubate at room temperature in the dark for 1-5 minutes. Different cells have different optimal culture times.
(3) After the incubation, centrifuge at 1000g for 5 minutes, remove the supernatant, add PBS and wash 2-3 times, 5 minutes each time.
(4) Use serum-free cell culture medium or PBS to resuspend the cells and observe them through fluorescence microscopy or flow cytometry.
2.2 Adherent cells
(1) Culture adherent cells on sterile coverslips.
(2) Remove the coverslip from the culture medium, suck out the excess culture medium, and place the coverslip in a humid environment.
(3) Add 100μL of Pyronin Y dye working solution from one corner of the coverslip, shake gently to evenly cover all cells with the dye, and incubate at room temperature in the dark for 1-5 minutes.
(4) Aspirate away the dye working solution, wash the coverslip 2 to 3 times with culture solution, and observe with a fluorescence microscope.
3. Microscope detection: The maximum excitation light of Pyronin Y is 540-550nm, and the maximum emission light is 560-580nm.

Notes:
① Pyronin Y is suitable for staining paraffin sections. It is recommended to prepare 5% Pyronin Y staining solution in PBS and incubate at room temperature in dark for 5-10 minutes;
② Fluorescent dyes all have quenching problems. Please try to avoid light as much as possible to slow down fluorescence quenching;
③ For your safety and health, please wear laboratory clothes and disposable gloves when operating.
References:
[1]. Taro Mannen, Tetsuro Hirose. RNase Sensitivity Screening for Nuclear Bodies with RNA Scaffolds in Mammalian Cells. 2017 Apr 20;7(8):e2232. doi: 10.21769/BioProtoc.2232.
[2]. Bo Li, Ying Wu, Xiao-Ming Gao. Pyronin Y as a fluorescent stain for paraffin sections. 2002 Jun-Jul;34(6-7):299-303. doi: 10.1023/a:1023325213198.