Hi, i think this pdf may help you

I have always used ultra cold acetone with the thinking that it would create less turbulence when mixing with water, less shear. That would seem like an easy side-by-side to perform. You can let us know.

When you say your plate is getting frosted, do you mean a plastic plate? Attach cells to glass slide/coverslip for acetone fixation.

It is a plastic plate with cell monolayer. Do I use acetone at room temperature for addition and then immediately keep the plate at 4 degrees for fixation to minimize evaporation and hence inhibit frosting ?

Acetone is not compatible with some plastics. It dissolves the plasticizers and makes the plastic non-transparent (probably what you are calling "frosting"). If you want to fix with acetone, you need to grow your monolayers on glass slides or cover slips. We do this by placing heat-sterilized Superfrost plus slides in a sterile square petri-dish. Some companies now sell dishes designed to take 4 slides in separate wells. We then take the slides out of the dishes and fix in a glass Coplin jar, thus there is no contact between plastic and acetone.

We also ultracool the acetone (I thought it was to remove any water).

Yes acetone can be kept in room temperature for fixation . No problem

Are you using a 100% Acetone? It doesn't matter RT or 4 degree, but you have to use 80% Acetone (in PBS) for fixation of cells attached on a plastic plate. For glass slides, it's ok using 100% Acetone. Good luck!

Thank you all so much. Dr. Feng Liu, Im going to try the using 80% acetone in PBS at 4 degrees. Hope I get my results this time without the problem of plate getting frosted along with my cells !! I will also try running a few more parallel experiments with 100% acetone,80% acetone in PBS and 80% acetone in distilled water and let you all know which gives a better result !!

You are welcome Sampada! For better practice, we usually put 80% Acetone in -20 degree untill use. After adding in acetone, plates can be left in RT for fixation (we do 10 min). It's interesting to see your comparison.

Grow cells on glass slides. Acetone will turn the plastics milky. Better fixative for clearer intracellular structures is 50:50 acetone with methanol at 4 degree C.

Use 80% cold ( kept at -20oC) acetone in PBS for 15 minutes and you will not have this problem anymore. See: H.B.C.R. Batista et al 2011Journal of Virological Methods Immunoperoxidase inhibitionassayfor rabies antibody detection

Thank you so much Dr.Roehe. I did try 80% Acetone in PBS as well as 80% acetone in deionized water, 100% acetone ( without discarding entire media in the well to avoid direct contact of acetone with adhered cells on the plate). All of them worked perfectly !!

However 80% acetone in deionized water and 100% acetone must be fixed for atleast 10 minutes at 4 C but for 80% acetone in PBS either room temperature or 4C for 10 minutes works !

Thank you all so much for the suggestions and advice. Appreciate it !!

No; keep it cool, otherwise the plastic will deteriorate! Regards

Yes Dr.Roehe , I store the 80% acetone at -20 C and add chilled 80% acetone to the plate . So I haven't experienced deterioration. But I do appreciate your suggestion, Thank you Dr.Roehe

hello,

we use -20°C stored 80% acetone (water) and fix at -20°C 30 min and always works.

no deterioration with plastic at room temperature (I've done the test with 100% and 80% acetone at RT : 100% deteriorates the plastic, not 80%)

regards

Chow is correct. acetone is likely melting the tissue culture plastic. Depending on your antigen you could try alternative fixitives such as 100% methanol or ethanol. I do not recommend paraformaldehyde since it can give high background. It is best if you can grow the cells on acid washed coverslips that you place in the culture dish after their sterilization. The added benefit is that you can use acetone and the cells will have higher definition and lower background when viewed under the microscope.

Good luck with your research

Thank you all so much... It has really helped me get through my experiments ! Cheers

:) wonrdefull