[Histonet] RE: IHC on brain cryosections... help!!

[C.M. van der Loos]

Hello Anna,
Besides all the good suggestions to your problem here is another one.
Since you are dealing with mouse tissue and you haven't said anything 
special about your secondary antibody I assume this was just an 
ordinary reagent. Usually these reagents are meant for application on 
human tissues and therefore adsorbed for human immunoglobulins, but not 
for mouse immunoglobulins. There might be a reaction of your secondary 
reagent with endogenous immunoglobulins in your tissue sections, 
causing the background staining as you described. If true, this 
background also occurs when leaving out your primary antibody from the 
protocol. You can abolish this background by mixing 10% normal mouse 
serum with your secondary reagent. You have to do this 30-60 min (room 
temp.) before actually applying it to the tissue section. 
Furthermore, you should reconsider the use of 3% hydrogen peroxide on a 
cryostat tissue section. If there are lots of erythrocytes around there 
will be an air-bubble formation that is damaging the tissue section. 
Instead, use 0.3% hydrogen peroxide + 0.1% sodium azide in PBS or TBS 
(20 min, RT). This is perhaps less efficient with respect to endogenous 
peroxidase in neutrophils but definitely more gentle to your tissue 
sections. 
Good luck!

Chris van der Loos, PhD
Dept. of Pathology
Academical Medical Center M2-230
Meibergdreef 9
NL-1105 AZ Amsterdam
The Netherlands

phone:  +31 20 5665631
fax:    +31 20 6960389
e-mail: c.m.vanderloos <@t> amc.uva.nl

----- Original Message ----- 
>From  "Anna Elisse Beaudin" <aep10 <@t> cornell.edu> 
Date  Tue, 7 Dec 2004 15:28:46 -0500 (EST) 
To  histonet <@t> lists.utsouthwestern.edu 
Subject  [Histonet] IHC on brain cryosections... help!! 


Hi all,

   I am having a TON of trouble with basic IHC on mouse brain cryostat
sections!  I have reviewed tons of protocols from the web and from
diferent papers.. and I am constantly getting confused between
protocols for fixed, paraffin-embedded sections, and those for
cryosections.    Here is an overview of the protocol I am currently
using:

-Collect cryosections on slides, leave at room temp 1-2 hours (as I'm
collecting)

- Quick-fix in buffered PFA (4%) for 10 minutes
- Rinse 3-10' PBS
- Block for endogenous peroxidase activity in 3% hydrogen peroxide in 
PBS
- Rinse 3 * 10 min Tris-buffered saline
- Block in 1%BSA, .3% triton X in TBS
- in primary o/n at 4 C
- Rinse 3 * 10 min TBS
- in secondary 1 hr at room temp
- Rinse 3* 10 min TBS
-- avitin-biotin (vector elite kit) for half hour at room temp
-- Rinse 2 * 10 min TBS, 1*10 min TB pH7.6
-- in DAB 2-3 minutes

With this protocol, I get terrible background in my negative controls, 
and
cannot identify any stain  in my positive slides.  The background I'm
getting does not appear to be cellular at all -- there is just brown 
gunk
everywhere.  Can anyone point me in the right direction?  Thank you so
much in advance for your help!

Best,
Anna Beaudin
Division of Nutritional Sciences
Cornell University
Ithaca, NY