ABC and brown color on the slide

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From:Tibor <> (by way of histonet)
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I  and my friend (both students) had to try out CK 5/6 at the hospital
where we will do our special project before finishing the last term at

We used the ABC technic with microwave antigen retrieval.
The procedure was like this:

We  dewaxed  the slides in xyleen for 2x10 minutes, in abs alc for 2x5
minutes   in   95%  alc  for  2x5 minutes and short time in 70% alc to
distilled water.

Before retrieval we blocked the endogenous peroxidase with 0.5 ml H2O2
in  50 ml Aq Dest in 30 minutes.

After retrieval in citrate buffer pH 6.0 we washed in PBS (pH 7.4) in 5 minutes
then we blocked with normal rabbit serum (1:5 in PBS) in 40 minutes.

The  primary  antibody  (monoclonal from Boeringer Mannheim Biochemica
cat.  no.  1273396)  was  diluted  in  1:20 and we incubated ON in the

The  day  after  we washed in PBS and applied the secondary biotinated
antibody (1:200 in PBS) and incubated at RM in 30 min.

The  ABC  was prepared taking 5 ml PBS 1 drop A and 1 drop B (from the
kit, sorry I forget to check which kit). We let the mixture stay on RT
in 30-60 minutes.

DAB  was  added  (first  we  washed  with  PBS) and we developed in 10
minutes.  After  this  time  we  washed  i  tap water in 5 minutes and
counter stained in Mayer's hematoxylin in 5 minutes and differentiated
with  1%  HCl in alcohol (this has a swedish name "saltsur sprit") and
then we did the bluing in tap water.

After  this  we  dehydrated  in  70% alc, 90% alc, 100% for 2x5 minutes and in
Xyleen 2x5 minutes and mounted in Mountex.

When our pathologist compared our slides with the ones done on Ventana
he found that the machine had better color (it uses Zn too).

Now,  our  problem  is that brown color on the slide around the tissue
(according  to  our pathologist we didn't had background in the tissue
itself).  We used PAN PEN, and we can see how this brown color appears
within the mark done with the pen. It's looking ugly...

I  hope  that I've provided enough information to get some suggestions
what to do. We are totally new and we never did this before.

Please, what we should do to get rid of that brown color on our slide?

In  march  we  have to do our project and then we are going to do much
more  slides.  It  wouldn't be fun having all of them brown around the

My friend and I would be very pleased to get any suggestions.

Thanks in advance!

Tibor Ric and Juan Paredes
students from Sweden (The University of Karlstad)

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