Author Topic: NG2 immunohistochemistry (Read 5027 times)

alportbury · « **on:** November 28, 2005, 02:49:22 PM »

Does anyone have any advice on how I can optimize NG2 staining in 10um fixed, frozen sections of adult mouse brain? I have been using Chemicon's anti-NG2 at 1:250 with routine 4% pfa fixation and have gotten terrible background - individual cell bodies are indestinguishable from background staining.
Is there another antibody that can be used for identifying oligo progenitor cells?

Thanks in advance for any help you can give me.
Andrea

« **on:** November 28, 2005, 02:49:22 PM »

Annica Andersson · « **Reply #1 on:** April 25, 2006, 10:24:10 AM »

Hello,
We been using NG2 (mouse) from chemicon 1:800 and develope with alexa-555. Works perfect!

alportbury · « **Reply #2 on:** April 25, 2006, 10:54:05 AM »

Thanks for the info. Are you using this antibody with 4% pfa fixed frozen sections? What thickness? Do you unmask?

Thanks so much,
Andrea

Annica Andersson · « **Reply #3 on:** April 26, 2006, 05:22:00 AM »

Hi,
We perfused the animal with 4%pf and picric acid then postfix for 90 min remove the brains to 10% sucrose and after 7 days we freeze the brains in isopentane, the brains are then cut in 12 um sections. We usually let the ab incubate for two days in 4C. No unmasking is needed.
I hope this will help you

alportbury · « **Reply #4 on:** April 26, 2006, 06:30:54 PM »

Thanks so much Annica, this is all very helpful.

Just 2 more questions:

* What % of picric acid do you use? Is it mixed in with the 4% pfa, kind of like Zamboni's fiaxative? Do you decolorize the tissue after fixation?

* Have you tried using the rabbit NG2 from Chemicon? If so, what were your results like?

O.K., that was more than 2 questions, but I really do appreciate your help with this.

Sincerely,
Andrea

ImmunoNYC · « **Reply #5 on:** April 26, 2006, 09:46:22 PM »

Hi Annica, Can you provide the catalog # of the antibody you find to work so well?

THANKS!

Annica Andersson · « **Reply #6 on:** April 27, 2006, 04:52:09 AM »

Hi,
We tried the rabbit ab from Chemicon but we got unspecific staining, the mouse is # Mab5384
We use a fixative that is called Lanas fixative
to make this you dissolve first the pf 40g/l in 0.4M Na2HPO4. 2H2O 288ml
heat to 58C when is clear you add 112ml 0.4M NaH2PO4.H2O and 140 ml saturated picric acid filter the solution and dilute with dH2O to 1000ml pH6.9.
No, we dont decolor the section after fixative.
good luck!

ImmunoNYC · « **Reply #7 on:** April 27, 2006, 10:19:39 PM »

Annica, Thanks for the information .... Do you use the MAB5384 on mouse tissue or rat? If it is mouse, how do you accomplish the mouse on mouse?

Thanks![/color]

Quote from: "Annica Andersson"

Hi,
We tried the rabbit ab from Chemicon but we got unspecific staining, the mouse is # Mab5384
We use a fixative that is called Lanas fixative
to make this you dissolve first the pf 40g/l in 0.4M Na2HPO4. 2H2O 288ml
heat to 58C when is clear you add 112ml 0.4M NaH2PO4.H2O and 140 ml saturated picric acid filter the solution and dilute with dH2O to 1000ml pH6.9.
No, we dont decolor the section after fixative.
good luck!

Annica Andersson · « **Reply #8 on:** April 28, 2006, 02:28:56 AM »

hi,
I have only tried it on rat.

« **Reply #8 on:** April 28, 2006, 02:28:56 AM »

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Author Topic: NG2 immunohistochemistry (Read 5027 times)