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:

http://www.myneurolab.com/mnl/mnlsite/ViewProduct.asp?idproduct=471001&c
atdesc=Histology+Equipment&subcatdesc=Sacrifice+Equipment&idsubcategory=
21

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.  
http://www.myneurolab.com/mnl/mnlsite/ViewProduct.asp?idproduct=476401&c
atdesc=Histology+Equipment&subcatdesc=Freezing+Devices&idsubcategory=187

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?

http://www.myneurolab.com/mnl/mnlsite/ViewProduct.asp?idproduct=064018&c
atdesc=Histology+Equipment&subcatdesc=Vibratory+Microtomes+%28Vibratome%
29&idsubcategory=181

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.


Cordially,

Charles W.  Scouten, Ph.D.
myNeuroLab.com
5918 Evergreen Blvd.
St. Louis, MO 63134
Ph: 314 522 0300  
FAX  314 522 0377
cwscouten@myneurolab.com
www.myneurolab.com


-----Original Message-----
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.

Dear all,

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 
cryoprotectant).

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-
075-148

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 
digress...

It's comforting to know I'm not alone.

Best wishes,

Andrew Gray
BPharm, BPharmSc(Hons)
PhD student
Melbourne