[Histonet] RE: IHC on brain cryosections... help!!
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
Chris van der Loos, PhD
Dept. of Pathology
Academical Medical Center M2-230
NL-1105 AZ Amsterdam
phone: +31 20 5665631
fax: +31 20 6960389
----- Original Message -----
>From "Anna Elisse Beaudin"
Date Tue, 7 Dec 2004 15:28:46 -0500 (EST)
Subject [Histonet] IHC on brain cryosections... help!!
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
-Collect cryosections on slides, leave at room temp 1-2 hours (as I'm
- Quick-fix in buffered PFA (4%) for 10 minutes
- Rinse 3-10' PBS
- Block for endogenous peroxidase activity in 3% hydrogen peroxide in
- 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,
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
everywhere. Can anyone point me in the right direction? Thank you so
much in advance for your help!
Division of Nutritional Sciences
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