Dear Tibor and friend

Three thoughts come to mind
1. This might be a washing problem. You do not mention how you went about
washing your slides: rinsing in a jet of PBS from a squishy bottle,  in a
coplin jar or in a staining dish of buffer with agitation. From personal
experiece I have found that washing in a dish with agitation to be the
better method, especially when soemthing like a PAN pen is used to isolate
the tissue. This seems to create a "well" in which the of the reagents tend
to stick around (due to their suface tension in a confined space - I think).

2. Check that the blocking serum  is  not cross reacting with that of the
seconadry biotinylated antibody (in the kit). We generally use the same
species ie if the secondary is raised in goat, then we block in goat serum.

3. Check the dilution of the Primary (unless you are using a pre-dilute)

Hope things work out - know the feeling

Best Regards

Louise Taylor
SAIMR
Johannesburg
South Africa




-----Original Message-----
From: Tibor [mailto:tibor@home.se]
Sent: 01 February 2000 07:21
To: histonet@pathology.swmed.edu
Subject: ABC and brown color on the slide


Hi!
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
university.

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
fridge.

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
tissue.

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)