[Histonet] RE: Automated immunostainer for research applications (Johnson, Teri)

Donna Harclerode dharclerode <@t> macropore.com
Wed Feb 23 19:49:05 CST 2005

   8. Automated immunostainer for research applications (Johnson, Teri)

Message: 8
Date: Wed, 23 Feb 2005 13:26:24 -0600

Hi all (and apologies to those on both lists),
For those of you doing research histology and immunohistochemistry,
which automated immunostainer do you use?  Do you do any overnight
incubations with it?  For those of you who have automated stainers and
formerly did overnight (4C) incubations, what modifications did you make
to your staining protocol to achieve the same result?  Also, do you
always use the substrate on instrument, or do you do that via
Some immunostainers require minimum amounts of primary antibody (~500
microliters), even if staining only one slide.  Some times we don't have
that much antibody to use.  Are there any tips to get around this with
the exception of manually pipetting as if doing a titration run?
Thanks for your input!

Teri Johnson
Managing Director Histology Facility
Stowers Institute for Medical Research
1000 E. 50th St.
Kansas City, Missouri  64110
tjj <@t> stowers-institute.org

Hi Terri

This is a bit more info than what I wrote on the other list

I use a "semi automated" system, the Shandon Sequenza.  I do research
staining and have not found any (to date) systems that would allow me
the flexibility to incubate at 4 oC overnight or some of the other
things I wanted to do. I run anywhere from 10-90+ slides at a time with
LSAB and DAB and it is no problem. 
The Sequenza system has a coverplate on one slide that keeps 80ul on the
slide.  The carriers make their own humidity chamber with 10 slides in
each carrier and I think they are great. I load the slides (after H2O2
blocking for paraffin), block with the Dako endogenous peroxidase block
for frozens, rinse in buffer and then primary etc. (I do not do a
separate protein block, just make up all the abs in Dako antibody
diluent and that has worked for many years for me) Once the slides are
loaded I just add the reagents (100ul) or buffer to the top.  
The only tricky part was learning to load the slides on the coverplate.
After the slides are loaded I always fill the reservoir with PBS to
check for misloaded slides.  If the slides are loaded correctly the
buffer drains slowly about 3-5 minutes.  If it drains too fast I know I
did not get the slide in correctly. I reuse the coverplates (they say of
course use only once) but as long as I only rinse them in DI water they
last about a month.  If you use soap they are ruined.   I use them for
frozen, cytospin and paraffin sections (paraffin usually are the
overnight ones. The other really nice thing is you can put tissue on
most of the slide and 100ul is plenty of antibody for each slide (I
always use 100 ul of antibody to be sure all the buffers are removed and
replaced by reagents). I do not DAB in the chambers, I remove the slides
for that so I can monitor the stain.  I have done fluorescent labeling
in the chambers and that has worked well. Anyhow the web info which is
I did not buy the holder for the racks, but just the slide racks and

Good luck with whatever you get and if you have any questions I forgot
to address, just let me know

Donna Harclerode, HT(ASCP)HTL, QIHC 
MacroPore Biosurgery, Inc
6740 Top Gun St.
San Diego, CA 92121
858-458-0900 ext 322
dharclerode <@t> macropore.com


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