Cell fixation with glutaraldehyde - (Dec/16/2009 )

Hi, I am doing a staining of my cells with crystal violet that get into de cell and binds DNA. The fisrt step is to fixate my cells with glutaraldehyde 1% and then stain them. The thing is that I cannot do the staining till next day, so I wonder what is the best way to preserve my cells after fixation, with/without glut. in water...

Thanks¡¡

Crystal violet binds to protein as well as DNA so the staining may not be specific, unless you have a DNA specific protocol!

I stain and fix the cells in one step for viability/kill curve assays using crystal violet in 0.4% glutaradehyde. You should be able to keep the cells in the glut solution for about a week at 4 deg C.

bob1 on Dec 21 2009, 12:59 AM said:

Vaginal Cytology - Cristal Violet Stain  

Reagent

Crystal violet 0.1% stain solution: dissolve 0.1 g of crystal violet powder to 100 ml of dH2O, mix well and store in a tightly sealed container at room temperature until needed.

Procedure

Place the mucous fluid on glass slide, and allow the smear to completely dry at room temperature. Once dry, these smears can be stained immediately or stored and stained at a later date.  Place the dry slide in a coplin jar (or other comparable staining vessel) containing the crystal violet, and stain for 1 min. Remove to a second coplin jar containing dH2O, and wash the slide for 1 min. Repeat once still this wash. Remove the excess dH2O from the edges of the slide with a light-duty tissue wiper, avoiding contact with the stained smear. Then pipette approximately 15 µl of glycerol on top of the smear and coverslip. Alternatively, other histological mounting reagents can be utilized to obtain a more permanent, non-diffusing stain. Examine the smear under light microscopy to determine cell types present. Microscopic examination should be done immediately after staining, because as the crystal violet will diffuse from the cells over time when using glycerol for coverslipping. Is easy to observe presence of cornified squamous epithelial cells, leukocytes, and/or nucleated epithelial cells.