RE: [Histonet] Histology of cell cultures
|From:||"Faucette, Lawrence (NIH/NIAID)" |
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
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
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
Disclaimer: The information in this e-mail and any of its attachments is
confidential and may contain sensitive information. It should not be used
by anyone who is not the original intended recipient. If you have received
this e-mail in error please inform the sender and delete it from your
mailbox or any other storage devices. The National Institute of Allergy and
Infectious Diseases (NIAID) shall not accept liability for any statement
made that are the sender's own and not expressly made on behalf of the NIAID
by one of its representatives.
Sent: Wednesday, September 15, 2004 10:24 AM
Subject: [Histonet] Histology of cell cultures
What methods are folks using to prepare histology preps of cell cultures?
Any help would be much appreciated/
Le présent message ainsi que ses éventuelles pièces jointes est
exclusivement destiné au(x) destinataire(s), personnes physiques ou
morales, qu'il désigne.
Il constitue de ce fait une correspondance à caractère privé et peut
contenir des informations confidentielles.
Si ce message vous est parvenu par erreur, nous vous remercions d'en aviser
immédiatement l'expéditeur par retour de courrier électronique puis de le
détruire, ainsi que ses éventuelles pièces jointes, sans en conserver de
This message, including any attachment, is intended for the use of the
individual or entity to which it is addressed.
It is therefore to be considered as a private correspondence which may
contain confidential information.
If you are not the intended recipient, please advise the sender immediately
by reply e.mail and delete this message and any attachment thereto without
retaining a copy.
Histonet mailing list
Histonet mailing list
<< Previous Message | Next Message >>