Did anybody use primary AB-anti-BrdU (VP-B209) from Vector?

[vivfly]

Hi, dear all: did anybody ever use the primary AB (anti-BrdU) from Vector lab? (VP-B209). I'm recently trying this AB for frozen sections. However, it never worked out. If you did successfully before,could you please share with me your experience? Thanks a lot!!!

[richard03]

You got no staining, background staining or else?

How did you pretreat the sections? Can you post the protocol you were using?

[vivfly]

Hi, richard03, thanks for your attention. recently i got some positive results by using high concentraion of the primary AB. However, it's accompanied by high background also. certainly, this high background could be improved by reducing the concentration of secondary AB (biotinylated anti-mouse, rabbit), but then it leads to less specific staining again. I would like to post my protocol briefly for discussion: Wash by PBS--fix by acetone(4degree) 15min--0.5%H2O2/MeOH incubation--permeabilize by 0.1%Triton X-100 15min--2N HCL incubation 30min (37degree)--0.5M NaHCO3 10min--50mM Tris buffer 10min--1%BSA/3%HS 30min--primary AB 1:200 2h RT--Secondary AB (biotinylated anti-mouse, rabbit)  1:1000 1h RT--ABC kit--develop.

[richard03]

Is this mouse tissue?

[vivfly]

Is this mouse tissue?

yes, it is. Did you ever use this antibody before? Any suggestions?Thanks

[richard03]

That is the problem.

Read this protocol, follow it strictly and you should be fine.

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

[vivfly]

Thanks a lot! I would like to try it!

[madeleine huey]

Hello,
You can try Zymed/Invitrogen's BrdU kit for mouse tissues.  I used it before, and no background what so ever (there's a special blocking solution in this kit to help reduced the background.  I tried different condition, and still find their kit is better than mine).
Good luck!
M