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Author Topic: No staining at all for IF.. Need advice to troubleshoot it.  (Read 6443 times)

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Offline SYeen

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No staining at all for IF.. Need advice to troubleshoot it.
« on: January 24, 2010, 08:01:39 AM »
Hello,

I am currently trying to do a single IF on cryostat brain sections by using an antibody which is supposedly to stain the protein on the plasma membrane of the astrocytes in the brain.However, after several attempts, I failed to obtain any result.I couldn't get a single fluorescence. The following is my protocol:

1. Immerse in PBS for 30 minutes
2. Block with the blocking solution which contains 5% donkey serum in PBS-0.1%TX for 1 hour in room temp
3. Primary antibody incubation (1:100) overnight in room temp
4. Wash with PBS for 3 times , 5 minutes interval between each wash
5. Secondary antibody (1:200) incubation in PBS for 1 hour in room temp
6. Wash with PBS for 4 times, 5 minutes interval between each wash
7. Apply coverslip

Since it is a plasma membrane protein, I did not wash the sections with PBS-Tx. I had also tried to use PBS-0.3% Tx, PBS-0.3% Tween, and PBS only in the blocking step (Step 2) instead of PBS-0.1%TX. Besides that, I also did antigen retrieval by using SDS in PBS. The protocol for the antigen retrieval was obtained from this website as well. Unfortunately, the results were the same. I am clueless and do not know how to troubleshoot my problem as I am a newbie in doing IF.Therefore, I am hoping that someone might be able to give me advice or enlighten me regarding this problem. Your help is deeply appreciated. Thanks.
« Last Edit: January 24, 2010, 08:04:46 AM by SYeen »

No staining at all for IF.. Need advice to troubleshoot it.
« on: January 24, 2010, 08:01:39 AM »

Offline excalibur

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Re: No staining at all for IF.. Need advice to troubleshoot it.
« Reply #1 on: January 24, 2010, 11:21:01 AM »
What is the species of your brain sections, host of primary antibody, and secondary?
Paula Keene Pierce, HTL(ASCP)HT
Excalibur Pathology, Inc.
5830 N Blue Lake Dr.
Norman, OK 73069
405-759-3959
www.excaliburpathology.com

Offline SYeen

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Re: No staining at all for IF.. Need advice to troubleshoot it.
« Reply #2 on: January 24, 2010, 08:39:43 PM »
The brain sections are from rat, the host of primary is rabbit and the secondary is donkey. Do you have any idea how should I troubleshoot it?

Offline CanuckPhD

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Re: No staining at all for IF.. Need advice to troubleshoot it.
« Reply #3 on: January 25, 2010, 02:52:36 AM »
How old are your antibodies? Have they been stored properly or gone through multiple freeze thaw cycles?

Check if your secondary works with another primary, preferably another astrocyte marker such as GFAP. Then you know if it is okay.

What astrocyte marker are you using? is there a reference for it working in IF or IHC? Are you using the same fixation conditions as the reference? Sometimes antibodies won't work in IF, no matter what you do.

Offline SYeen

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Re: No staining at all for IF.. Need advice to troubleshoot it.
« Reply #4 on: January 25, 2010, 08:41:00 AM »
I just bought my antibody few months ago (~3 months) and I just opened the vial 3 weeks ago.I have made aliquots of the antibody and stored it in -20 degrees as stated in the datasheet of the antibody. Or do you think I should store it in -4 degrees instead?

My secondary works well with GFAP, which I used to stained sections from the same rat brain.

Actually my plan is to double label the this antibody(target) with GFAP(marker). However, I couldnt even get a single stain for the target and hence, I couldnt proceed to the double labeling procedure.

In the datasheet for the target antibody, it is stated that immunohistochemistry and IF was not tested on this antibody before. However, I have tried using this antibody to do IHC on perfused rat brain sections and I managed to get decent staining.

So are u suggesting that this problem of mine could be due to the primary or secondary antibody used?

Offline excalibur

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Re: No staining at all for IF.. Need advice to troubleshoot it.
« Reply #5 on: January 25, 2010, 11:30:14 AM »
If your primary is hosted in rabbit, the secondary should attach to the rabbit and state so.
Paula Keene Pierce, HTL(ASCP)HT
Excalibur Pathology, Inc.
5830 N Blue Lake Dr.
Norman, OK 73069
405-759-3959
www.excaliburpathology.com

Offline SYeen

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Re: No staining at all for IF.. Need advice to troubleshoot it.
« Reply #6 on: January 25, 2010, 10:03:36 PM »
The secondary is donkey anti-rabbit antibody. So is there any suggestion that you can give me to troubleshoot it?

In your previous reply, you mentioned that some antibodies don't work in IF. Are you refering to the primary antibody? So some antibodies may work well in other assays but maybe not in IF?
« Last Edit: January 25, 2010, 11:02:23 PM by SYeen »

Offline voland

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Re: No staining at all for IF.. Need advice to troubleshoot it.
« Reply #7 on: January 26, 2010, 03:46:14 AM »
Are the sections from PFA perfused animals? If not you should do fixation first?

Offline Cardio

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Re: No staining at all for IF.. Need advice to troubleshoot it.
« Reply #8 on: January 26, 2010, 08:50:35 AM »
The secondary is donkey anti-rabbit antibody. So is there any suggestion that you can give me to troubleshoot it?

In your previous reply, you mentioned that some antibodies don't work in IF. Are you refering to the primary antibody? So some antibodies may work well in other assays but maybe not in IF?

Exactly. Some ab's are able to have multiple application while others are only good for one. Its not wise to buy ab's that have not been tested for the application that you want to use it for. I would suggest contacting technical support and see if they have any pub's for the ab's. Sometimes they are listed on the product sheet and sometimes they are not.

You can try a antigen retrieval and see if that helps.

Offline SYeen

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Re: No staining at all for IF.. Need advice to troubleshoot it.
« Reply #9 on: January 26, 2010, 08:55:30 AM »
Are the sections from PFA perfused animals? If not you should do fixation first?

Yes, I perfused the rats with PFA 1st before i proceed on the IF.

Offline SYeen

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Re: No staining at all for IF.. Need advice to troubleshoot it.
« Reply #10 on: January 26, 2010, 08:58:55 AM »
The secondary is donkey anti-rabbit antibody. So is there any suggestion that you can give me to troubleshoot it?

In your previous reply, you mentioned that some antibodies don't work in IF. Are you refering to the primary antibody? So some antibodies may work well in other assays but maybe not in IF?

Exactly. Some ab's are able to have multiple application while others are only good for one. Its not wise to buy ab's that have not been tested for the application that you want to use it for. I would suggest contacting technical support and see if they have any pub's for the ab's. Sometimes they are listed on the product sheet and sometimes they are not.

You can try a antigen retrieval and see if that helps.


Okay, thanks for your suggestion. The antibody has been tested on western blot and ELISA only as stated in the datasheet.  I have found a publication using the same antibody for IHC and it works fine. I would not have thought that this antibody has problem with IF.

I have already done antigen retrieval in my last attempt using SDS method as found in this website but failed to get any result. Is there anything I can do besides contacting technical support?

Offline Cardio

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Re: No staining at all for IF.. Need advice to troubleshoot it.
« Reply #11 on: January 26, 2010, 10:28:28 AM »
You can try the citrate antigen retrieval.

Its a pretty common one and not all antigen retrievals give the same results.

Re: No staining at all for IF.. Need advice to troubleshoot it.
« Reply #11 on: January 26, 2010, 10:28:28 AM »