This document is a summary of message
from the Purdue University Cytometry Laboratories Cytometry Discussion list.
Fixation issues Submitted by
"Kennedy, E. (Elaine)" E.Kennedy@organon.co.uk on 12 February 2003 22:52
Hi Elaine
I will reply
directly to you instead of the list because this may
sound like a
commercial endorsement but it is not - Caltag's Fix and
Perm is the
best. I have used it on human and mouse
PB and BM and on
many cell
lines with many monoclonal antibodies and it always works,
maintains the
scatter patterns well, and does not result in much cell
loss. It is a 2-step procedure with a 15 minute
incubation and a
wash step
between the 2 reagents.
As in any new
procedure, you will have to try it out with your application.
--
Barbara J Taylor
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Elaine,
In my
experience it is true that you may have to try several methods until you find
the optimal technique. However, in
human cells we have found that Caltag's Fix & Perm Kit works almost every
time. Rarely have I had a problem using
this.
(Oh - and it's
really easy! Way easier than ethanol
fixation!)
Good Luck!
Abby Kelliher
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Hi Elaine!
The most
simple and in most cases perfect fixation procedure for
intracellular
proteins is using cold 70% Ethanol. It works for most
proteins, you
can store your samples in the fridge for a year or longer
without any
changes in antigen distribution, you can do a simultaneous DNA analysis for
cell cycle etc. Due to the possibility of long time storage you can analyse all
the samples from an experiment at the same time which improves comparability.
If your protein is very small there may be a problem with loosing it, although
most, even small proteins are precipitated. There have been some cases
described in the literature that the conformation of some antigens may change
so that the antibodies will not recognize them anymore, but these cases are
very rare. The only thing you have to take care of is the possible formation of
aggregates during the fixation procedure. Here is the protocol I use for
intracellular immunofluorescence:
Centrifuge
your cells in a tube with a conical bottom, discard the
supernatant, resuspend the cells completely in the
remaining drop. Now
comes the
tricky part: Put the tube on a vortex machine, keep it shaking while adding the
ethanol dropwise and slowly. This is to avoid aggregate formation. Use ~1ml
ethanol for 5x10e6 cells. Keep in the fridge until use.
Before
staining centrifuge again, discard supernatant, resuspend cells in remaining
droplet. Add 1ml Buffer (0.1%Tris.HCl, 0.1% Triton X100, 2mM MgCl2 pH7.4) the
same way as when you added the ethanol (dropwise and slowly while vortexing).
Wash again with the same buffer, again taking care to avoid aggregates. Wash
again with trisbuffer including 20% FCS or 2% BSA and incubate for 30 min to
block unspecific binding sites. Wash again blocking buffer and include your
antibodies. After the last staining step was with buffer w/o FCS or BSA and
analyse.
Hope this
helps.
With kind
regards,
Nicole
--------------------------------------------------------------------------------------------------
Hi Elaine,
I can
recommend CALTAG Fix and Perm for intracellular staining and I
have used this
product in both diagnostic and research laboratories.
The reagents
are easy to use and also allow concurrent surface
staining. The
FSC/SSC presentation does not alter significantly
compared to
other permeabilising reagents.
I have no commercial
interest in CALTAG products and I offer this
information as
my opinion only.
Good luck!
Cathy
------------------------------------------------------------------------------------------------
Elaine,
In my
experience you may need to look at different fixatives as well as
permeabilization
agents.
We are looking
at human white blood cells and we need both good
intracellular
perm as well as good light scatter properties (for differentiating the WBC
subsets). This may not be a concern for you since you're using a cell line.
If you're
looking at something novel intracellularly, use something known first to
validate your fix/perm . For example, with our
WBC work we
look for good myeloperoxidase labeling in granulocytes to
validate the
fix/perm.
I hope this
helps.
Peter
Peter Lopez
-----------------------------------------------------------------------------------------------------------------------
Hi Elaine,
Ethanol is a
penetrating fixative and conditions of use
including cell type will govern the distance of effect into a cell, for
most cells it is a total fixation.
For DNA
staining with dyes this is fine but for Ab you have to know they are against
the fixed epitope.
For most
Ab used in Flow Cytometry it is
preferable to Fix the cell membrane with paraformaldehyde then use a detergent
to permiabilise said memebrane.
FIX
Paraformaldehyde
fixative solution PFM
4 % (w/v) paraformaldehyde
3 % (w/v) sucrose ( I never used sucrose in my fixation mix
only in the Hypertonic solution
desined for membrane
shedding to recover nucli. You
could try it for comparison)
in PBS pH 7.4.
Store at 4°C in the dark for a few
days.
PERM
Triton
permeating solution
1 % (v/v) Triton-X100
in PBS pH 7.4.
Store at room temperature for a few weeks.
Saponine
permeating solution
0.1 % w/v
in PBS pH 7.4.
Store at 4°C for a few days.
Nonident
permeating solution
0.5 % (v/v) Nonidet P-40
in PBS pH 7.4.
Store at 4°C for few days.
N-OctylGlucosamine (NOG) permeating solution
0.74 mg/l (w/v) NOG (This is the
Critical Micelle
Concentration for this
detergent)
in PBS pH 7.4.
Store at 4°C for few days.
The problems
with " home brew" are reproduction, QC and shelf life so I went to a
commercial product. We sell the IntraPrep kit for this which has a PFM
fixation and Saponin Perm. 50 test
IM2388 and 150 test IM2389 As a positive control to demonstrate perm and
staining we have anti-Tubulin conjugated with FITC part 6607113
Regards
Martin
-----------------------------------------------------------------------------------------------
Hi Elane,
I did some
intracellular staining in thymocytes some time ago. I fix my
cells with
formaldehy solution.
My experience
was that is was critical, how fresh the formaldehyd solution was. I took 2%
formaldehy and end up with 1% end concentration for fixation.
I attach a
protocol in pdf format and hope this helps a bit
Good luck
Steffen
====================
Dr. Steffen
Schmitt
----------------------------------------------------------------------------------------------------------
Elaine,
I have used
the Caltag fix and Perm kit for staining of intercellular
parasites with
SOME luck, but I had better luck with 70% MeOH (Never
tried
EtOH). I would appreciate hearing
relpies you get to this because I will likely try again soon.
Mike
-----------------------------------------------------------------------------------------------------------
Elaine,
We've actually
pondered this same question at Cell Signaling since our
customers use
a variety of different fix&perm methods.
We have settled on a protocol that involves fixation in 1% formaldehyde
for 10 min at 37C, then permeabilization in 90% methanol. This protocol works very well for every
antibody we tried. Some of our
collaborators recently compared this protocol to a saponin-based fix&perm
kit from Invitrogen and screened with a number of antibodies. All of the antibodies worked well with the
aldehyde/methanol, but over half of them did not work at all with the
saponin-based kit. The only down-side
to methanol permeabilization is that the scatter characteristics of the cells
are not as clear, so it may be difficult to pick out different cell types in a
heterogeneous suspension on a scatter plot.
This isn't an issue for researchers like you that are working with cell
lines.
For a detailed
copy of our protocol, please visit our website
(http://www.cellsignal.com),
click on Support, then Research Protocols, and finally on Flow. Please feel free to contact me if you have
any questions.
Best of luck,
--Randy Wetzel
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