[Histonet] Re 17. fluorescence immunohistochemistry

Donna Harclerode dharclerode <@t> cytoritx.com
Tue Dec 20 15:06:17 CST 2005

Message: 17
Date: Mon, 19 Dec 2005 22:08:48 +0100
From: Emily.Wiesner <@t> medecine.unige.ch
Subject: [Histonet] fluorescence immunohistochemistry
To: histonet <@t> lists.utsouthwestern.edu
Message-ID: <1135026528.43a72160c086f <@t> webmail.medecine.unige.ch>
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Hi All,
I am new to fluorescence immunohistochemistry and I was wondering if
anyone can let me know the simple steps involved in performing this
technique. I know the dilutions required for the primary and secondary
antibodies, I just need to known what solutions are used for washes and
the steps in between!
Thanks in advance,

Dear Emily

I do very simple IHC using fluorescent secondaries from Jackson
Immunoresearch and purified primary abs.  Depending on what you are
doing, there can be more steps added.

I usually am staining sections on a slide or cells on chamber slides or
in a plate.

For the unfixed frozen sections on slides I normally would fix 5 min in
75% acetone 25% ethanol and then right into PBS- do not allow the fixed
slides to dry.  The only change would be if the antibody prefers
formalin fixation to acetone/ alcohol (thanks Gayle- I love this
fixative for almost everything).

I load them into a Sequenza(tm) rack (Thermo Electron, formerly Shandon)
or you could put them in a humid chamber of some sort, wiping excess
buffer before adding antibody or blocking solutions.

OPTIONAL I have recently started using Image-iT(tm) FX signal enhancer,
I36933, $80 Invitrogen for auto fluorescence (not sure it helps yet, but
sure does not hurt) 30 minutes.  I would not use this on routinely- my
sections have infracted areas that have been a problem in the past)

After blocking, I rinse 1 time in the Sequenza rack (3 x 5 minutes if
you are doing them by hand)

Add 100ul of the antibody diluted in Dako antibody diluent S0809-
usually 1:100- 1:1k (dilutions are antibody dependant, but I have found
in fluorescence too high a concentration is not as big a problem as with
HRP formats)
If you are using a humid chamber be sure to cover all the tissue.

I have incubated slides at Room temperature for 1-2 hours, but now am
staining overnight in the fridge (not so good in clinical situations)

After primary incubation, I rinse 1 time in the Sequenza rack (3 x 5
minutes if you are doing them by hand)

Next is Jackson secondary at a 1:100 dilution (I use Cy3 conjugates for
single color and FITC and RITC for dual color. All my secondaries are
minimal cross (mouse to rat), but if all your tissue is human that is
not necessary.  30 minutes 

I add DAPI to my secondaries to give a blue nuclear stain so I can tell
what I am looking at and localize the stain.  

Rinse again 3 times in PBS and coverslip with your choice of aqueous
media.  For media that never gets hard, I prefer Immunomount (Thermo
Electron, formerly Shandon) or Aqua Mount (Lerner from VWR) same thing
different label or the Prolonge Mounting media P36930 for $101 that will
harden. I do not like the hard mount media from Vector- I got many air
bubbles forming day after the sldes were coverslipped.

For cells on plates or chamber slides I usually fix with 10% NBF or 4%
PFA (same concentration of fixative, 4%PFA will contain no methanol) for
5-30 minutes.  CD makers can be very hard to stain with NBF but with a
short fix, I have been pretty luck so far. I rinse 3 x 10 minutes with

I have not used the blocking reagents on cells as I have not seen any
autofluorescence in our cardiac myocytes, neurons or stem cells.

The rest of the procedure is the same except in a 6 well plate you need
a lot of antibody to cover the plate (about 600u).  I try to use a
rocker if it is available to help to be sure of good coverage.

I do not usually cover my slides or plates, but if your room is very
bright or your signal very week, it would be a good idea to protect the
secondaries from light.

I like to use isotype controls (negative controls) or rabbit IgGs for
much of my work, but in a pinch I use just the antibody diluent to be
sure the stain I see, is a result of the primary antibody and not the
secondary binding to some part of the cell. In cell assays I always want
to stain a known positive and a known negative cell if at all possible
in addition to the isotype controls.

If you are making your own diluent (to me Dako diluent is great and I
have used it successfully for over 10 years in many applications) 
I use 0.3% Triton, 5 % Normal serum from the species that your secondary
is made in - (all my secondaries are made in donkey so I would use
donkey serum) in PBS.  I have used donkey secondaries when possible
because when I tested them years ago, they were a bit cleaner on the CD
markers I was using.
Dako diluent must have a similar make up, but it lasts a year in the
fridge and it permeablizes intact cells very nicely. 

One big thing when purchasing fluorophores, make sure the filters on
your scope can see the secondaries you are purchasing.  I found out at
my current job after staining with Cy3 and RITC that the reason I could
not get any stain was they had a Texas red filter not the Cy3, RITC
filter.  Ideally if you are doing multicolor you want narrow band
filters so you can separate the colors, with broad pass filters the
colors blur together and it is hard to separate the results.

Good Luck!


Donna Harclerode, HT, (ASCP), HTL, QIHC
Scientist / Immunohistochemistry
Cytori Therapeutics
3020 Callan Rd.
San Diego, CA 92121
858-458-0900 ext 322
dharclerode <@t> cytoritx.com

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