intracellular staining
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
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 formaldehyde solution.
My experience was that is was critical, how fresh the
formaldehyd solution was. I took 2% formaldehyde 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,
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 saponinbased 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
CLB protocol for membrane and intracellular FACS staining : (By
Paul Baars, CLB-KVI) Reagents: PBS PBS 0.5% BSA PBS 0.1%
saponin 0.5% BSA Human Pooled Serum (HPS) 4% Paraformaldehyde
(PFA) in PBS Procedure (the whole procedure is performed on
ice)
1. Wash the cells in a 15 ml tube in PBS 0.5% BSA
2. Suspend the cells in PBS 0.5% BSA (4x106/ml) and add direct
conjugated Mab's for the membrane staining
3. Incubate for 30 min
4. Wash 1X with PBS 0.5% BSA
5. Wash 1X with PBS
6. Add 1.5 ml 4% PFA in PBS and incubate for 5 min (stopwatch!!)
7. Wash 1X with PBS
8. Wash 1X with PBS 0.1% saponin 0.5% BSA
9. Suspend the cells in PBS 0.1% saponin 0.5% BSA + 10% HPS
10. Incubate for 20 min
11. Wash 1X with PBS 0.1% saponin 0.5% BSA
12. (From now on this buffer is used until the end of the
procedure)
13. Suspend the cells in a x 50ml (a= number of different
intracellular staining) and pipette the cells in a 96-well dish
14. Add Mab's to the intracellular antigen and control Mab's
(diluted in saponin buffer)
15. Incubate for 30 min
16. Wash 3X 17. Measure the cells on the FACS We know that
other permeabilization procedures are also successful for
Granzyme staining e.g. the BFA fixation,
john voorn
For B27 as a PE or APC conjugate, we typically use 1 mcg/ml
final concentration. We titer every lot.
I don't know what the
stock concentration of your B27 mAb is, so if you want the exact
calculation you will have to do the calculation or send me the
concentration offline. A guess is that the stock concentration
is 200 mcg/ml (100 mcg in 500 uL) and you are using it at a
1:285 dilution, which gives a final concentration of 0.7 mcg/ml.
Looks pretty reasonable to me. A common mistake that is further
propagated by many of the Ab companies is to use antibodies by
mass as for example, requiring "0.5 mcg per test". The variable
that makes the difference is mAb concentration, not total
amount. We typically stain in 50 ul and thus "per test" only
requite 1/4 the total mass of mAb to maintain the same
concentration as you would when staining in 200 ul.
Calman Prussin Allergic Diseases Section NIAID/ NIH
Although I agree monensin is not the perfect blocker of
intracellular transport, I think your response is a bit
exaggerated. What toxicity data are you citing? The Jung paper
demonstrated monensin toxicity only after 16 hours. Both
monensin and BFA need only be in the culture for 2-4 hours for
maximal effect (my data and others). Also consider that the
strong stimuli (PMA/ionomycin) that are often used to effect
sufficient cytokine expression are also causing cell death. A
couple of hours of monensin may not be the largest perturbation
in the system. I have not compared a large number of cytokines,
but for huIL-2 and IFN I do not see a significant difference in
the mean fluorescence intensity or % positives between BFA and
monensin. The main reason for using monensin in the past was
that it was dramatically cheaper. Sigma now sells BFA at a
reasonable price. It is worth having a through discussion of
the relative merits of each, as I am eager to switch to a
better reagent. I look forward to hearing from you to
substantiate your statement. Intracellularly Backed Up in
Bethesda,
Calman Prussin Allergic Diseases Section NIAID/ NIH
The protocol that I find most useful for permeabilisation for
the detection of internal antigens is to use saponin. It is
relatively gentle (ie it doesn't put enormous holes in the
cytoplasm or the cell membrane) and can be used in conjunction
with surface staining or DNA staining. You may have to play
about with conditions but a good starting point is to treat
cells with 0.3% saponin for about 15mins at room temperature
before doing the antigen detection and then to use 0.1% saponin
in all subsequent steps. Be careful with the washing steps as
it is easy to lose all your cells! We have found that the
permeabilisation is best at room temperature and that the
saoponin needs to be present all the time as the
permeabilisation effect can be reversed. As with all
intracellular staining washing is important, but if you are
using directly conjugated antibody, this reduces the steps and
reduces cell loss. Hope that this is of some use.
Derek Davies Imperial Cancer Research Fund London
Hi Dr. Roy and fellow flowers: I do this all the time. I have
abandoned Western blot in favor of intracellular flow cytometry,
and I have compared indirect to direct staining, with similar
results. I use either a biotinylated first step or an unlabeled
first step. If I use an unlabeled, I use a ligand-affinitypurified polyclonal, and a labeled F(ab2') for the second step.
I block with Ig of the same species as the F(ab2'). I have
successfully seen p38, STAT3, JAK2, and a lot of cytokines. The
first step does not have to be one specifically for flow to
work--if it works in westerns and immunohistochemistry chances
are excellent that the Ab will work here too. A major pitfall is
not blocking sufficiently and not washing sufficiently. I block
with several mg/ml Ig for about 1 hr on ice after
permeabilization. Also, the first wash after adding Ig, Ab, or
second step must be with buffer added and left there for the
same length of time as the incubation step. For example, if you
left Ab on for 45 min, you must spin out the Ab and then leave
the wash buffer on for 45 min, then proceed with the subsequent
washes as usual. Another thing I found was that Caltag fix &
permeabilization reagents gave me superior signal to noise
ratios for phospho-Ab labeling, while Pharmingen or home-made
reagents gave me very good results for intracellular cytokines.
See these references for more details: Fleisher et al., Clin
Immunol 90:425-430, 1999 Barton & Murphy Cytokine 12:18-27, 2000
Feel free to contact me for more info. Best,
Beverly Barton
Assistant Professor Dept. of Surgery