RE: UPDATE: Immunohistochemistry is making me cry.
|From:||"Charles W. Scouten, Ph.D." |
Some ideas that may help you. You should be using the Perfusion One
apparatus and protocol to do the perfusion, which will avoid soft tissue
shrinkage and distortion and give better quality cell preservation.
Gravity is not enough pressure (unless you work in a cathedral), and
peristaltic pump controls the wrong parameter. See the link below:
Brain freezing would go better with the Clini-RF. Nitrogen is too cold,
it causes the Macro cracks. But fluid immersion in a very cold liquid
is the only way to really avoid the swiss cheese if you must freeze.
Instead of freezing, many people avoid a lot of problems by cutting with
a Vibratome(tm), and get much better preservation of cell membranes and
hence better staining of cytosol proteins. The drawback is you can't
cut thin sections, under 20 microns, but you don't want to anyway. Can
you borrow a Vibratome(tm) from a user in your shop to try?
Thick sections can be cut in a cryostat, I have done it many times, up
to 60 microns, just double click the advance lever. Your Venetian
blinds were due to cutting the tissue too cold. After nitrogen or other
supercold quick freezing, the block needs time, maybe an hour, in the
cryostat to gently come up to cutting temperature.
The staining I leave to other histonet experts.
Charles W. Scouten, Ph.D.
5918 Evergreen Blvd.
St. Louis, MO 63134
Ph: 314 522 0300
FAX 314 522 0377
From: Andrew Gray [mailto:Andrew.Gray@vcp.monash.edu.au]
Sent: Tuesday, June 10, 2003 4:30 AM
To: HistoNet Server
Subject: UPDATE: Immunohistochemistry is making me cry.
It seems as though I struck some kind of resonance on the list
yesterday; thank you for your kind responses. I will give a brief
account of what I have been up to (aka 'The Saga'). I hope this does
not come across as self-pity.
My aim has been to do immunofluorescent co-labelling studies of the mu
opioid receptor in the rat CNS. The problem has been poor mu
staining. I established IHC operations in my department virtually
single-handedly (which have since flourished!), and sought out
expertise from external institutions, including much from the
HistoNet. I had done some DAB and IF IHC in skin during my honours
year, which were simple enough.
I had been told by some auto-rad people that brain freezing by CO2
pellets would be "fine". It was not, and I went through the 'swiss
cheese' rite of passage that all CNS IHC people go through (freezing
too slow). So I spent a couple of weeks getting messy with crushed
CO2, liquid nitrogen, isopropane, sucrose and OCT. I discovered that
perfusion of the rat was required, so I learnt the surgery behind that
(again from outside) and constructed a few perfusion apparatuses before
getting it right. A couple more weeks and that was sorted out.
Then I had problems with macroscopic tissue cracks (tissue too large,
freezing too rapid), and microscopic tissue cracks (these went away
mysteriously, may have had something to do with too much time in
I was told thick sections (50um) were inappropriate for a cryostat,
after initially suffering 'venetian blind' artifact and tissues
breaking apart. The problems were solved after playing with cryostat
temperature (lucky, because we don't have a freezing microtome), and
using yet smaller tissue pieces and embedding in OCT.
Then, uneven autofluorescence was a problem (uneven distribution of
Vectashield mountant, I think).
Then, poor staining. Attempts to resolve this included using slide-
mounted sections, RT and 4 deg C incubations, shakers, Citrate buffer
antigen retrieval (microwave and hot plate), light irradiation to
reduce autofluorescence, narrow-band microscope optics and varying
incubation times. All to little avail.
I have been able to achieve excellent immunoreactivity with my other
Ab's, but not the mu one. The antigen levels should be about the same
(abundant). I have been able to obtain weak mu specific staining at
the lowest dilution attempted (1:500) but it is not strong enough to
unambiguously determine IR in individual cells.
Unfortunately the co-localisation aspect is an absolute linchpin of my
work; this is an all or nothing situation. To add insult to injury,
the mu Ab works brilliantly well in the spinal cord and fairly well in
the myenteric plexus.
My next move is to add Normal Goat Serum to my diluent. This may seem
like a default component of a Ab diluting medium to many; I had been
dissuaded from ordering NGS by a staff member, who disputed its
benefit. They suggested that 2% rat serum would suffice. The rat
serum has helped, a little. Also, varying the Triton-X100 level
(currently 0.1%) is on the cards.
My antibodies are:
Primary: Chemicon Guinea-pig anti-mu opioid receptor-1 polyclonal
antibody Cat. AB1774
Secondary: Jackson Texas Red Donkey Anti-Guinea Pig IgG (H+L) Code 706-
I should add that I had applied for an extension to my scholarship
yesterday, which had me feeling a bit like a criminal. But I
It's comforting to know I'm not alone.
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