Protocols for Hoechst Staining

The Hoechst stains are fluorescent dyes used for labelling DNA and RNA through fluorescence microscopy and flow cytometry techniques. Hoechst 33258 and Hoechst 33342 are the most used of these stains. They have the ability to bind to DNA molecules, causing the nuclei and mitochondria to appear fluorescent under ultraviolet light. Protocols for Hoechst staining are common practice in biotechnological and genetics laboratories, and include cell staining techniques using both Hoechst 33258 and Hoechst 33342.

Hoechst 33342 HSC Staining and Stem Cell Purification Protocol

• This protocol is used to stain cells from mice bone marrow. Technicians prepare bone marrow sample with HBSS+ salt solution, HEPES buffer and fetal calf serum. They spin the solution and add to a pre-warmed DMEM medium. Hoechst stain is then added. The solution is placed in a 98.6 degrees Fahrenheit water bath for exactly 90 minutes, reports the Department of Immunology at the University of Pittsburgh. DNA segments appear fluorescent under a cytometer.

Hoechst 33342 DNA Staining

• During this protocol, technicians suspended Hoechst 33342 stain in distilled water, add 2 percent fetal calf serum to the cell sample, keeping the pH at 7.2 with the help of a buffer solution. Cells are then incubated at 98.6 degrees F for an hour, and immediately analyzed with a piece of equipment called cytometer.

  Stained DNA is then visible and fluorescent, according to the University of Michigan Health System.

DNA Detection Using the DNA-binding Fluorochrome Hoechst 33258

• This protocols used a piece of equipment called spectrophotometer to analyze DNA and RNA segments. Technicians use Hoechst 33258 working solution and calf thymus DNA samples. They also make a separated solution using the DNA sample and a chemical called ethidium bromide. Later, they compare the cell staining results in both media, using a spectrophotometer, according to Current Protocols in Cytometry.