Yongzhong Wang and interested others:

In our experience, if plates are not air dried prior to fixation and again prior to staining, cells detach from the plate leaving you nothing to stain. This may merely be a peculiarity of our bone cell cultures. Our osteoblast precursor cell cultures (CFU-F) are much more adherent and less likely to float away. Osteoclast cells however, are not inclined to stick to the plastic dishes. They much prefer bone slices. Staining bone slices is rather straightforward. You'll have to experiment with your particular cells to see if air drying is needed and where. Our entire procedure manual is rather long, but I'm having it converted into a PDF file and placed on our website. When that process is complete, I'll inform the histonet ether. In the meanwhile, I can email pertinent portions to interested parties as a word document.

Regards, Donna Montague
University of Arkansas for Medical Sciences
Departments of Physiology & Orthopaedic Surgery
4301 W. Markham St. # 505
Little Rock, AR 72205
(501) 603-1239

-----Original Message-----
From: Yongzhong Wang [mailto:Yongzhong.Wang@tufts.edu] 
Sent: Thursday, January 30, 2003 7:26 PM
To: Montague, Donna C
Subject: RE: Staining cells


Donna,
Would you please send me the procedure by email? Right now I am doing 
something similar and an having some problems in my staining. I don't 
understand why you need to air dry completely before fixation and before 
staining. Will the cells still be intact that way?
Thanks,
William Y Wang,
Tufts Univ
Dept. of Chem & Biolo Engr
ywang09@coral.tufts.edu

Quoting "Montague, Donna C" :

> SeonaCherie and interested others:
>  
> I have procedures for all but Masson's on dishes. In general you can 
> stain cells as you would sections but keep in mind a few things: After 
> you have removed the media from the cells allow them to air dry 
> completely before fixation. Apply the fixative gently from a pipette 
> using half the volume of the well (e.g. 12 well plate each well 
> generally holds 2 mL of media, so you can apply 1 mL of fixative). 
> After they have fixed (usually 15 - 30 minutes at room temp is 
> sufficient), dump the fixative off the dishes, rinse by immersion in a 
> shallow container that is being filled with gently running water. The 
> only way I know how to do this is one dish at a time. Allow the plates 
> to air dry before staining.
>  
> I can fax, mail or email the procedures to you. Your call. Let me 
> know. Donna Montague University of Arkansas for Medical Sciences
> Departments of Physiology & Orthopaedic Surgery
> 4301 W. Markham St. # 505
> Little Rock, AR 72205
> (501) 603-1239 
> 
> -----Original Message-----
> From: SeonaCherie Hansen [mailto:hanse150@umn.edu]
> Sent: Thursday, January 30, 2003 11:25 AM
> To: histonet@pathology.swmed
> Subject: Staining cells
> 
> 
> Does anyone out there have a procedure for staining cultured cells in 
> the culture dish? We will be doing an Alizrin Red, an O Red O, Masson 
> Trichrome and Alkaline Phosphotase.
>  
> Thank you,
>  
> SeonaCherie
> 
>