The rats are sacrificed and transcardially perfused with saline followed with 4% PFA (pH 7.4). Then the brain was removed and post fixed in 4% PFA overnight, followed with 30% sucrose until SINK.
We cut the brains with 25 um or 40 um (specially for BrdU staining) on cryostat. The sections are mounted to geltain-coated slides and dried up.
1. Rehydration: PBS 10 min
2. Citrate buffer: 95 degree, 30 min
3. PBS 2 X 5 min
4. 2N HCl 37 degree, 30 min
5. Borate Buffer 2 X 10 min
6. PBS 5 min
7. 1% BSA, 0.3% Triton, 10% Goat Serum, 1:1000 Rat-BrdU (Abcam) overnight (for double staining just include other mouse source antibody here)
1% BSA, 0.3% Triton, 10% Goat Serum, 1:500 mouse-BrdU (for double staining have to perform another staining after this)*
8. PBS 3 x 5 min
9. 1: 200 Goat-anti-Rat-488 in PBS 1.5 hr
10. PBS 3 x 5 min
11. DAPI 1:10K 2 min
12. PBS 5 min
*For double staing with another mouse-source antibody, from
11. Mouse anti-NeuN (for example), 1:1000, overnight
12. PBS 3 x 5 min
13. 1:200 Goat anti Mouse 568 in PBS 1.5 hr
14: PBS 3 x 5 min
15. DAPI 1:10K 2 min
16. PBS 5 min
发件人： Gayle Callis
发送时间： 2008-07-15 07:03:26
主题： Re: [Histonet] Reply double immunostaining on same species, mouse
This was the previous messages we exhanged, and there is no mention of what your secondary antibodies were???? I would be interested in seeing what your exact protocol is???? Also, PFA may be affecting the outcome of your staining. There is no mention of species being stained either.
----- Original Message -----
To: Gayle Callis ; Histonet
Sent: Sunday, July 13, 2008 8:06 PM
Subject: Re: [Histonet] Reply double immunostaining on same species, mouse
That's cool and please kindly let me know the detail.
Yes we used PFA to fix tissue and we have both microtome / cryostat to make sections.
the cryostat sections are embeded with O.C.T.
We have several mouse monoclonal antibodies, and I will use only Alexa fluroscence conjugated 2nd antibody rather DAB staining.
Thanks a lot.
发件人： Gayle Callis
发送时间： 2008-07-14 07:05:57
主题： [Histonet] Reply double immunostaining on same species, mouse
We do double and triple immunfluorescence staining on mouse tissues with all antibodies raised in same host species only on mouse tissues, rat antiMouse. It takes some careful blocking and organization, but it works beautifully. Please contact me and I will discuss the details on how to set up the staining, but supply some details on what you want to see as green or red fluorescence. This is only done on frozen sections and NOT on formalin fixed paraffin embedded tissue to eliminate autofluorescence. There are several ways to set this up, and we do use some biotinylated primary antibodies along with Molecular Probes Streptavidin Alexa fluor dyes. Gayle M. CallisHTL/HT/MT(ASCP)Bozeman MT You can still have issues with the second red detection trying to stick to the first AB unless you do some very aggressive blocking.
Sent: Saturday, July 12, 2008 9:58 AM
Subject: [Histonet] Double immunostaining with two same source 1st Antibody
Just wonder any one of you tried to perform IHC using two primary antibodies from same source, mice, for example?
The process is that
Mice anti-A (1st IHC)->secondary antibody labeled with GREEN
-> Mice anti-B (2nd IHC)-> Red fluroscence
then there's no cross reaction.