Abcam Ad

Author Topic: MOM blocking  (Read 11705 times)

0 Members and 1 Guest are viewing this topic.

Offline madeleine huey

  • SilverMember
  • *****
  • Posts: 56
MOM blocking
« on: August 25, 2005, 09:04:17 PM »
Hello,
H.....elp!!!!  
Anyone has a detail protocol/s for mouse ab on mouse tissue blocking?  
I don't want to use commercial kit (doing huge amount IHC).  
Madeleine   :cry:

MOM blocking
« on: August 25, 2005, 09:04:17 PM »

Offline richard03

  • Administrator
  • GoldMember
  • *****
  • Posts: 1212
MOM blocking
« Reply #1 on: August 26, 2005, 09:48:33 AM »
Madeleine,

I have used unconjudgated anti-mouse IgG to block endogenous mouse IgG with some good results but stability is an issue. I am trying to stablize my method using Fab fragment anti-mouse IgG and I think it should work better.

I will post my detailed protocol here if it was successful in next couple of weeks.

Richard

Offline madeleine huey

  • SilverMember
  • *****
  • Posts: 56
MOM blocking
« Reply #2 on: August 26, 2005, 01:05:40 PM »
Great!  Couldn't wait to hear good news from your out come next week.  The  high background is adding more grey hair on my head.

I am thinking about this stragedy in the meantime.  By mixing primary antibody (already optimize in-house 1st ab =1ug/ml)  with secondary antibody (already optimize; Alexa 488 Goat anti-mouse @ 1:1000) at 4C for overnight. Next day add 1 ug/ml normal mouse serum to the overnight  mixture for 2 hours, and use this ab mixture for my mouse tissue (normal mouse serum + 1st Ab IgG + conjugated 2nd Ab).  Do you think this stragedy will work (gather this idea from some ref.)?

Thanks again!
Madeleine

Offline richard03

  • Administrator
  • GoldMember
  • *****
  • Posts: 1212
MOM blocking
« Reply #3 on: August 26, 2005, 01:34:30 PM »
Good thought! I have played this ONCE (normal mouse serum + 1st Ab IgG + conjugated 2nd Ab) around over a year ago WITHOUT much success. It was very likely (I thought) that all the primary antibody binding sites covered by secondary antibody so there is no or little "open binding sites" for the primary ab to bind to its tissue antigens. In addition, the mixture makes bigger molecules therefore penetration is greatly reduced.

However, don't be discouraged by my words. Just give it a try. I think the key here is the ratio of primary ab and secondary ab. Also make sure when you incubate the mixture, it should be in concentrated form or close to concentrated form. Then dilute the mixture to appropriate concentration and apply it to tissue sections. Longer incubation time may be needed.

Richard

Offline ImmunoNYC

  • Global Moderator
  • GoldMember
  • *****
  • Posts: 684
MOM blocking
« Reply #4 on: August 29, 2005, 11:03:06 PM »
Precomplexing totally works but needs to be optimized. I normally try varying amounts of primary and secondary to see what works best (from 1-10ug/ml in various ratios).

However, I would suggest instead of using mouse serum the next day you used purified mouse IgG to block.

That is what I do and it works for most antibodies - especially those which you know work under normal circumstances.

Offline richard03

  • Administrator
  • GoldMember
  • *****
  • Posts: 1212
MOM blocking
« Reply #5 on: August 30, 2005, 09:11:56 AM »
Hi MaximinaNYC,

Can you post a little more details about the Precomplexing method? I have tried this method once without any staining. I also tried M.O.M kit from Vector Labs and it works but can not eliminate background completely. I am trying to use unconjugated Fab fragment anti-mouse to block endogenous mouse IgG and have not got any satisfactory results yet.

Richard

Offline madeleine huey

  • SilverMember
  • *****
  • Posts: 56
MOM blocking
« Reply #6 on: August 30, 2005, 08:58:48 PM »
Thank-you MaximinaNYC,
We would love to see your detail protocol!
Madeleine

Offline ImmunoNYC

  • Global Moderator
  • GoldMember
  • *****
  • Posts: 684
MOM blocking
« Reply #7 on: August 30, 2005, 10:33:22 PM »
Sure, I have it on my other computer and will post it when I get to work. I gave it out at the NYSHS meeting this past May for working with human antibodies on human samples.

It should work with most antibodies but definitely works better or easier when the antibody is "robust" meaning that it is a easy-to-use working antibody. I have used it on human and mouse samples with success - although I must admit I still prefer to biotinylate or otherwise label my primaries if possible to avoid MOM or HOH staining entirely.

Offline richard03

  • Administrator
  • GoldMember
  • *****
  • Posts: 1212
MOM blocking
« Reply #8 on: August 30, 2005, 11:16:52 PM »
Thanks MaximinaNYC. I also found a article dealing with "pre-mix" method. I haven't got chance to read it through. I will post the article when I get to work tomorrow.

A good news is that my Fab fragment anti-mouse blocking method seemed to work fine. Now I need a final confirmation if I had time this week, and fine tune the protocol. I will let you guys know if I got it to work.

Richard

Offline richard03

  • Administrator
  • GoldMember
  • *****
  • Posts: 1212
MOM blocking
« Reply #9 on: August 31, 2005, 09:09:02 AM »
Here is the article I found about "pre-mix" method

Article

Offline madeleine huey

  • SilverMember
  • *****
  • Posts: 56
MOM blocking
« Reply #10 on: August 31, 2005, 01:21:52 PM »
Hello MaximinaNYC,
Can you give us more information on your biotinylation kit (i.e Vendor name, cat. #, etc....).  Or did you have your own protocol for labelling your ab with biotin reagent?  
Detail protocol would be greatly appreciated!
Madeleine

Offline ImmunoNYC

  • Global Moderator
  • GoldMember
  • *****
  • Posts: 684
MOM blocking
« Reply #11 on: September 01, 2005, 09:43:38 AM »
I get my biotinylation kit from Molecular Probes. Verrrrrry easy. Other times I use biotinylation NHS reagent from Pierce and also Pierce sephadex columns.

Offline madeleine huey

  • SilverMember
  • *****
  • Posts: 56
MOM blocking
« Reply #12 on: September 02, 2005, 06:45:07 PM »
Hello Richard,
I have just finished my ihc with 20 ug/ml purified goat anti-mouse F(ab) blocking @ RT for 1 hr.  The background still exist.  Did you have a better luck with you Fab blocking yet?  I would like to see how you did it.
Here's my protocol;
1) cut 10 um pre-fix 4% PFA frozen section & dry @ 50C for 30'
2) hydrate 3x TBS
3) block Avidin/Biotin for 60 min each
4) 3x washing with TBS-T
5) block Non-specific with 20ug/ml goat.anti-mouse Fab for 1 hr
6) 3x washing with TBS-T
7) incubate with 1st ab/10%NGS+1%BSA+0.5%Tx100+0.02%NaN3+TBS for overnigh @ 4C
8) 3x washing with TBS-T
9) quench with 0.3% H202/MeOH for 60 min
10) 3x washing with TBS-T
11) Incubate 2nd ab (Dako's Envision plus; Polymer goat anti-mouse) for 30 min
12) 3x washing with TBS-T
13) DAB+ for 1 min (already strong brown color)
14) 3x washing with TBS-T
15) Counterstain
16) washing with TBS-T
17) hydrate & mount with Permount

Offline madeleine huey

  • SilverMember
  • *****
  • Posts: 56
MOM blocking
« Reply #13 on: September 02, 2005, 07:10:55 PM »
Hello MaximinaNYC,
Don't forget to post your detail "pre-mix" protocol for all of us.
Happy Labor Day!
Madeleine

Offline richard03

  • Administrator
  • GoldMember
  • *****
  • Posts: 1212
MOM blocking
« Reply #14 on: September 03, 2005, 09:44:33 AM »
Madeleine, MaximinaNYC and All,

Finally!!! - I made it.

After 3 years struggling with this mouse on mouse thing, I finally got it to work and it works so beautifully that I could not believe myself it is true.

The detailed protocol was just published on IHC World Main Site and here is the link:

http://www.ihcworld.com/_protocols/general_IHC/immuno_mom.htm

Although this protocol works well, but I would like anyone who uses this protocol to report here so I can improve this protocol from time to time. Thus other researchers will be benefitting from your contributions as well.  

Please feel free to post your comments, suggestions, experience after using this protocol.

Wish all the best and have a wonderful labor day weekend!

Richard

MOM blocking
« Reply #14 on: September 03, 2005, 09:44:33 AM »