Fixation and IHC thanks and add'l info

From:"Allen, Steven"

Thanks to everyone who replied to my query about fixation time and IHC
(original post below).  I didn't go into too much detail about my procedure
for the sake of brevity, but there's been several questions so I'll try to
answer them as best as I can.  I'm very new to IHC and I'm basically doing
this alone.

Our necropsy unit usually fixes tissue in 10% formalin for at least 24-48
hours at RT.  The tissues are trimmed and usually processed by Histology the
same day and embedded the following day.  I suppose I could arrange for a
shorter fixation time for future tissues, but I'm stuck with the tissues I
have now.    

All slides for IHC are mounted on Probe-on plus slides and IHC is performed
using MicroProbe holders.  I am currently testing 3 antibodies (S100 mouse
monoclonal, S100A6 rabbit polyclonal, and CD34 mouse monoclonal).  S100 has
been reported positive in astrocytes, so I tried rat brain sections.  I also
purchased "positive control slides" from one of the vendors (human tonsil).
For CD34, I have tried rat kidney and a human tumor cell line grown in nude
rat lung.  I have tried two dilutions 1:50 and 1:100.  I am getting some
background staining but no real specific staining.  The "positive control
slides" are negative.

One vendor recommends antigen retrieval and another recommends trypsin
digest, so I did both.  Did not realize I might be killing the epitopes by
doing both.  I do my antigen retrieval by placing the slides into coplin
jars and microwaving for 10 minutes using step-down power (100%-4 min, 60%-1
min, 40%-1 min, 30%-4 min).  30 minute cool down. I am not familiar with any
other methods for antigen retrieval.  I can easily try a different antigen
retrieval solution (EDTA, Tris, Zinc sulfate) and I probably will.

All my reagents are less than 6 months old.  I have had positive results
with p53, p21, MDM2 and excellent results with vimentin.  I have not
obtained positive results with any of these 3 new antibodies.  Once I
optimize a procedure, I have a set of 47 rat tumors to subject to IHC.

Sorry for the long winded post.  Original post is below.
Thanks, -Steve Allen-
Good morning,
I am doing some IHC on rat tissues (multiple antibodies-one at a time).  Due
to poor results with several antibodies, I have been speaking with technical
support and they have recommended a formalin fixation time of anywhere
between 2 and 12 hours for tissues intended for future IHC.

Our necropsy unit usually waits a minimum of 24 to 48 hours before trimming
and embedding.  I cannot go back and undo the fixation time for my tissues,
I have to use them.  I realize the problem of cross-linking with extended
formalin fixation.  I am performing antigen retrieval: 0.01M citrate (pH
6.0), 10 min. microwave boil.  I am also performing a 0.16% trypsin digest
(30 min) before the blocking step. 
I have obtained positive results with other antibodies, using the same
tissues.  Also, my tissue blocks are about 4 years old.

Could/should I extend the boil time for antigen retrieval?  What kind of
fixation times do you use for IHC tissues?
Thanks in advance,

Steve Allen
Research Technologist
Lovelace Respiratory Research Institute
Albuquerque, NM

<< Previous Message | Next Message >>