[Histonet] Background in frozen pancreas sections (continued...)

Martin S. sonya.martin <@t> soton.ac.uk
Fri Mar 2 08:00:44 CST 2007

I followed your advice - Avidin/Biotin block, serum in the antibody
diluents and preincubation of the goat anti-rat and I got some great
staining with no background - Thanks!

I don't know if it was all down the avidin/biotin rather than SA/biotin
but I have had problems with the SA/B block before on other tissues
giving more background than when I left it out.

I use the ImmunoPure Peroxidase Suppressor from Pierce Biotech, its
pricey but very convenient and you only need a little on each section.
I've used it on mouse spleen, lymph node, pancreas and thyroid and human
lymph node and tumours with excellent results.

Thanks again for your help,


-----Original Message-----
From: Andrea Hooper [mailto:anh2006 <@t> med.cornell.edu] 
Sent: 20 February 2007 17:07
To: Martin S.
Subject: RE: RE: [Histonet] Background in frozen pancreas sections

Interesting. My lab has had nothing but problems with the
Streptavidin/Biotin blocking kit. Vector claims not to know what we are
talking about but the background was SO MUCH WORSE using that kit,
whereas when we changed to the Avidin/Biotin kit all background

Also about Vector secondaries ... I hate them! I never use anything but

Without using the anti-rat IgG, mouse adsorbed from JAckson I think you
will have difficulty getting rid of the background (though
preaincubating with mouse serum or better yet pure mouse IgG is a good
suggestion and may work). My recommendation is to invest the $100 and
buy the mouse adsorbed donkey anti-rat IgG.

Just looking over your protocol ... try blocking with 5% BSA in
adddition to goat serum and use 1% BSA in all your antibody steps. I
also hope you dilute your primary/secondary in some goat serum also.
This will prevent your primary from binding to your blocking reagents
(use 2% serum).

Finally I have no experience with peroxidase suppressor ... is this
stuff good?

Good luck, let us know how it works out!

>It was the Streptavidin/Biotin block I was using from Vector. Also, now

>that I look at the antibodies again the goat anti-rat from Jackson 
>doesn't seem to be mouse adsorbed - however I did try a rabbit anti-rat

>from Vector which was mouse adsorbed and the background was worse.
>I also tried fixing with 95% ethanol straight after sections were cut 
>(instead of acetone) but saw no inprovement.
>I've attached a few images to show you what I'm talking about - also in

>the image x10-1 do you know what the mass of cells on the left is - 
>could it be a lymph node do you think?
>Next time I will try with the avidin/biotin block, longer washes and 
>try the mouse adsorbed anti-rat and also the goat anti-rat 
>pre-incubated in NMS for 1hr (as someone else suggested) - I'll let you

>know how I get on!
>Heres my protocol;
>- Cut sections (10um) and air-dry 30mins
>- Fix in 100% Acetone, 10min (room temp)
>- Air-dry briefly and wash with PBS
>- Block with 10% normal goat serum in PBS, 30min
>- Wash PBSx3
>- Block with Streptavidin, 15min (Vector Streptavidin/Biotin blocking
>- Wash PBS, 3x5min
>- Block with Biotin, 15min
>- Wash PBS, 3x5min
>- Incubate with rat antiCD8 (BD Pahrmingen; diluted to ~5ug/ml), 2hr 
>(room temp)
>- Wash PBSx3
>- Incubate with goat anti-rat:Biotin, 45min (Jackson; 
>biotin-SP-conjugated AffiniPure F(ab)2 fragment; product number 
>112-066-003; diluted 1/1000, ~1ug/ml in 10% normal mouse serum in PBS)
>- Wash PBSx3
>- Apply peroxidase suppressor, 15min (Pierce)
>- Wash PBSx3
>- Apply StreptABCcomplex, 45min (Vector)
>- Wash PBSx3
>- Apply DAB, 2min (Sigma)
>- Wash PBSx3
>- Wash tap water
>- Counterstain with haematoxylin
>-----Original Message-----
>From: Andrea Hooper [mailto:anh2006 <@t> med.cornell.edu]
>Sent: 19 February 2007 17:52
>To: Martin S.
>Subject: Re: RE: [Histonet] Background in frozen pancreas sections
>I took this off Histonet for now ....
>How long were your incubations in avidin then biotin? Which kit 
>specifically did you use? How long were your washes? When I used the 
>streptavidin biotin blocking kit from Vector I found my background was 
>always worse and now only use avidin biotin blocking kits from Vector.
>15 min in Avidin, 2 x 10 min washes, 15 min in biotin, 2 x 10 min 
>As far as why you get background if you are using Jackson's mouse 
>adsorbed anti-rat ... I don't get it. How did you fix your samples?
>Also, what concentration of primary, secondary and strep-HRP are you 
>using? What is the catalog # of the antibody from Jackson?
>I stain frozen pancreas all the time and get little background so I 
>want to help you figure this out :)
>----- Original Message -----
>From: "Martin S." <sonya.martin <@t> soton.ac.uk>
>Date: Monday, February 19, 2007 11:31 am
>Subject: RE: [Histonet] Background in frozen pancreas sections
>>  Thanks Andrea,
>>  My goat anti-rat is from Jackson (F(ab)2, mouse adsorbed) but I find

>> that if I don't dilute it in 10% normal mouse serum I get a lot of  
>> background - diluted in this way it seems clean on spleen sections 
>> but
>>  maybe its different for pancreas.
>>  I used Vector's avidin/biotin blocking kit after serum block but  
>> beforeprimary - I will try this again concentrating on the washes.
>>  Thanks again
>>  Sonya
>>  -----Original Message-----
>>  From: Andrea Hooper [anh2006 <@t> med.cornell.edu]
>>  Sent: 19 February 2007 13:55
>>  To: Martin S.
>>  Cc: histonet <@t> lists.utsouthwestern.edu
>>  Subject: Re: [Histonet] Background in frozen pancreas sections
>>  (continued...)
>>  It's likely your secondary (anti-rat IgG) is cross reacting with 
>> mouse
>>  IgG in the tissue. I would suggest you purchase an anti-rat IgG 
>> which  is highly cross adsorbed to many species INCLUDING mouse. This

>> can be  purchased from Jackson ImmunoResearch for example.
>>  Also, with pancreas you definitely need to do an AB block. You said 
>> it
>>  increased background? That's odd and concerning ... how are you 
>> using  the kit? Lots of washes? I perform my AB block specifically 
>> after  serumblock and before primary (with loads of washing) and am 
>> able to  get very little background.
>>  ----- Original Message -----
>>  From: "Martin S." <sonya.martin <@t> soton.ac.uk>
>>  Date: Monday, February 19, 2007 5:28 am
>>  Subject: [Histonet] Background in frozen pancreas sections
>>  (continued...)
>>  > Sorry just thought some additional info might be useful;  >  > The

>> pancreas tissue was frozen in OCT in a bath of isopentane on  dry  > 
>> ice, 10um sections were cut, air dried for 1hr, then fixed in  
>> acetone  > for 10mins. I quenched endogenous peroxidase with Pierce  
>> Peroxidase  > Suppressor.
>>  > Sections without primary or secondary antibodies still have a  
>> littel  > background but its worse with the secondary (I have tried 
>> rabbit  > anti-rat:Biotin and it was worse).
>>  >
>>  > Ta
>>  >
>>  >
>>  >
>>  > _______________________________________________
>>  > Histonet mailing list
>>  > Histonet <@t> lists.utsouthwestern.edu
>>  > http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>>  >
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