[Histonet] Histology of cell cultures

Wed Sep 15 10:29:39 CDT 2004

This is the protocol we have been using for years.  Hope it helps

Live cell preparation. 
Prepare 1% Agarose, cell culture grade, in iso osmotic PBS. You need to boil
to dissolve it in PBS. Check for loss of water as vapor during boiling and
replace with distilled water. 

Bring to approx 50 C°. The agar will solidify below this point. 

Prepare your cells as a cell suspension in minimal amount of medium, at room
temperature. 

To make a 0.5 ml agar block you may need between 10 to 20x106 cells. You can
scale this down to as little as 0.5 x 106 in 50 µl but your cells will be
very sparse on section. 

Prepare in advance 1.5 ml Eppendorf tubes (or small PCR tubes for
micro-preparations) to which you cut off and discarded the conical bottom.
Cap the tube. 

Mix evenly and thoroughly your cells with 0.5 ml of agar (or less) and very
quickly transfer to the capped inverted tube. No bubbles.  

Place the still molten agar and tube on ice until is solid. 

Then open carefully the cap, and gently extract the agar cylinder from its
base (the cap). At this point you can cut the agar piece in two with a razor
blade, freeze half and fix in formalin the other. Your cells are still alive
and biochemically active at this point. 

Fixed cell preparation. 
Proceed as above, but you fix the cells before. Then you can use Agarose in
PBS, regardless of osmolarity. 

Fixing the cells before embedding, prevents movement of labile antigens or
loss of short-lived molecules.

Lawrence J Faucette

Contractor

HT ASCP

http://www.niaid.nih.gov/dir/services/animalcare/VetPathology/VetPathology-i
ndex.html

Infectious Disease Pathogenesis Section

Comparative Medicine Branch 

Division of Intramural Research, NIAID, NIH

Twinbrook III, Room 2W-01A, MSC 8135

12735 Twinbrook Parkway

Bethesda, MD 20892-8135

Telephone 301-451-1056

Fax 301-480-2343

SoBran, Inc.

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-----Original Message-----
From: Stephen.Eyres <@t> sanofi-synthelabo.com
[mailto:Stephen.Eyres <@t> sanofi-synthelabo.com] 
Sent: Wednesday, September 15, 2004 10:24 AM
To: Histonet
Subject: [Histonet] Histology of cell cultures

Hi All,

What methods are folks using to prepare histology preps of cell cultures?
Any help would be much appreciated/

Cheers
Steve

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