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Author Topic: BrdU IHC - Loss of protein?  (Read 3453 times)

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Offline WeAre138

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BrdU IHC - Loss of protein?
« on: March 02, 2011, 12:16:13 PM »
Well, I feel as if I only post here when I need help, but I'm still pretty new at this game and don't feel like any advice I have would be much help to anybody.

I've been working with BrdU IHCs and am having some issues I was hoping someone here might be able to help me with.  It seems to me that a lot of cellular protein is getting degraded in the processing (probably Trypsin?), but I could be wrong.

Relevant protocol steps: 3% H2O2 10min; Trypsin:PBS (1:10) 30min @37C; 2N HCl 30min @37C

All images are C57 adult control testis exposed to 100mg/kg BrdU 1.5h prior to sacrifice.

10X with no counter stain:


10X with hematoxylin counter stain:


40X with no counter stain:


40X with hematoxylin counter stain:


Hopefully someone here can share their knowledge.  Thanks.

BrdU IHC - Loss of protein?
« on: March 02, 2011, 12:16:13 PM »

Offline MT Scientist

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Re: BrdU IHC - Loss of protein?
« Reply #1 on: March 02, 2011, 12:40:51 PM »
What kind of slides are you using?

Offline WeAre138

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Re: BrdU IHC - Loss of protein?
« Reply #2 on: March 02, 2011, 02:06:56 PM »
Thermo, positive charge.

Offline MT Scientist

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Re: BrdU IHC - Loss of protein?
« Reply #3 on: March 02, 2011, 03:14:00 PM »
I did BRDU way too long ago.
Looking at a random BRDU staining protocol, you may need to tweak the times for trypsin and HCl (10min and 15 minutes respectively in that protocol).  That may be the cause of your tissue loss - too much retrieval....
« Last Edit: March 02, 2011, 03:33:18 PM by MT Scientist »

Offline Cardio

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Re: BrdU IHC - Loss of protein?
« Reply #4 on: March 03, 2011, 10:45:31 AM »
I would bet my house on it.

Offline WeAre138

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Re: BrdU IHC - Loss of protein?
« Reply #5 on: March 03, 2011, 03:05:17 PM »
@MT - That's interesting, I looked at 4 protocols when I wrote up my protocol and none of them used trypsin or HCl for less than 20 minutes.  But, looking at my sections, it's a bit too long.

Thanks for all the help folks, I really appreciate it.

Re: BrdU IHC - Loss of protein?
« Reply #5 on: March 03, 2011, 03:05:17 PM »