Fixing might play a role but more importantly the type of slides used may effect the adhesion of the tissue onto the slide.

You will need to troubleshoot. Insufficient attachment or insufficient fixation would seem like the two possibilities. How do you attach and how do you fix? Do you know what is in the Sigma NBF and if it is still good?

I agree with Dr. Tchokonte-Nana. 10 min in 10% NBF may be enough to fixing cells. From my experience with bone tissue, that dettachment of some portions of tissue/cells are common when uncoated glass slides are used.

The problem was solved by using poy-L-lysine coated glass slides,

I am not sure exactly what is the protocol used by you (it may be a good idea to share all the steps performed).

However, based on my evaluation you are not performing the protocol right. As cytopathologists we process many types of specimens with cells in the clinical samples. In light of this you cannot make cytology preparation from formalin fixed cells.

I am recommending following two options for oil red o staining-

I. Better option is prepare a smear of your cell suspension like preparing a peripheral blood smear, wet fix it (by putting the slide in 95% alcohol before the smear dries on the smear). Then stain this smear as follows:

i. Rinse with 60% isopropanol

ii. Stain with freshly prepared Oil Red O working solution 15 mins

iii. Rinse with 60% isopropanol

iv. Lightly stain nuclei with alum haematoxylin 5 dips

v. Rinse with distilled water

vi. Mount in aqueous mountant or glycerine jelly.

II. Other option is to study as a wet preparation (mix the cell suspension and oil red o and make a wet preparation by putting a drop on a slide and putting a coverslip on it. Then see the wet preparation under microscope with condenser lowered down as done to evaluate wet preparations.

Reagents (copy-pasted from http://www.ihcworld.com/_protocols/special_stains/oil_red_o_ellis.htm

1. Oil red O stock stain

oil red O (CI 26125) 0.5 g

isopropanol 100.0 ml

Dissolve the dye in the isopropanol, using the very gentle heat of a water bath. This is the stock stain.

2. Oil Red O working solution

For use: Dilute 30 ml of the stock stain with 20 ml of distilled water, allow to stand for 10 mins, and filter into a Coplin jar, and cover immediately. The stain does not keep, and should be made up fresh from the stock solution each time.

3. Glycerine Jelly Mounting Medium

Gelatin (Kitchen grade) 10 g

Distilled Water 60 ml

Glycerol 70 ml

Phenol 0.25 g

Dissolve the gelatine in the distilled water using sufficient heat to melt the gelatine, add the glycerol and phenol. Mix well and transfer to a small capped bottle and refrigerate.

May be depends of the slides used and the poor adhesion produce artifacts of the tissue

Rohini, have you had this problem before or is this the first time? From past experience (over 30 yrs ago!) with rat hepatocyte cultures, coated slides were essential to ensure cellular attachment. I did try poly-L-lysine and that helped but I still had cellular detachments. The adhesive protein that really worked for me for Fibronectin. I really don't think the cause was due to fixation duration as 10 minutes should be long enough to cross-link the proteins. Good luck!

It is certainly not a problem of fixation but of the glass slides used. Put the glass slide into ether to de-fat them or use specially prepared glass slides. Some loss of cells will always occur during the staining processes. This is especially annoying in specimens from fluids containing often few cells like urine. In such cases we prefer spray fixation.

In fact the slides air dried stain well with Leishman types of stains where the cells are fixed during the staining procedure itself without loss of any material or imparting the bald spots.So sloughing of the cells doesnot appear to be a function of ill-fixation.

I think 10 minutes fixation long enough to cross-link the proteins. This problem may be depends of the uncoated glass slides are used.

. good luck.

Common problem when using uncoated slides. You can decrease the degree of peeling of cells if poy-L-lysine or APES coated glass slides are used.