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Author Topic: IF is working but IHC doesn't work, is it possible?  (Read 9884 times)

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Offline apple

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IF is working but IHC doesn't work, is it possible?
« on: December 07, 2005, 02:25:35 PM »
Hi,

I have done some staining for cell slides. For immunofluorescent method it's working but it does work for immunocytochemistry method. Why? Is it possible? Usually IHC is more sensitive than IF?
Have you met same problem?

Thanks

IF is working but IHC doesn't work, is it possible?
« on: December 07, 2005, 02:25:35 PM »

Offline ImmunoNYC

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Re: IF is working but IHC doesn't work, is it possible?
« Reply #1 on: December 07, 2005, 09:43:02 PM »
Can you give us more details of the protocol specifically? I can't really help otherwise!

Quote from: "apple"
Hi,

I have done some staining for cell slides. For immunofluorescent method it's working but it does work for immunocytochemistry method. Why? Is it possible? Usually IHC is more sensitive than IF?
Have you met same problem?

Thanks

Offline apple

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IF is working but IHC doesn't work, is it possible?
« Reply #2 on: December 08, 2005, 08:09:35 AM »
Hi,

My IF protocol is as following:

1. Take slides out from -80C.
2. Air dry for 30min
3. Fix in -20C acetone 10min
4. wash in cold PBS+0.5% BSA + 2mM EDTA
5. block with 0.1% BSA in PBS for 30min
6. apply primary ab (Biotin labelled) 1hr at 4C (primary ab is diluted in PBS+1% BSA)
7. wash 3 times in wash buffer
8. apply FITC (avidin labelled) 40min RT
9. wash 3 times in wash buffer
10. apply PI nucleus counterstain
11. observe.
All the procedure was done in cold (on ice or in fridge)

My ICC protocol is:
1. take slide out from -80C
2. leave for 10min at RT
3. fixed in -20C methanol and permeabilized in -20C acetone for 1min
4. wash 3 times in buffer (cold PBS+0.5% BSA + 2mM EDTA)
5. block with 1% BSA in PBS FOR 20min RT
6. wash 3 times in buffer
7. apply primary ab 1hr RT
8. wash 3 times
9. apply ready to used peroxidase Avidin 30min RT
10. wash 3 times
11. apply DAB for 4 min
12. wash in tap water
13. couterstain with Mayer's Haematoxylin 4min
14. wash in tap water

My results are strange. IF works but ICC doesn't work...... Why??

Offline ImmunoNYC

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IF is working but IHC doesn't work, is it possible?
« Reply #3 on: December 08, 2005, 09:15:33 AM »
I see that you use acetone for one procedure and methanol for the other. The one that isn't working is the one with methanol. I never used methanol for CD markers. Perhaps that is the problem?

How about if they are the same cell preparations in both procedures? Or different?

Are you sure your staining is specific? You only block with 0.1% BSA in the first protocol, doesn't seem like enough to me. Why do you block woth 0.1% BSA and then incubate primary in 1% BSA. That doesn't make a lot of sense.

Are you using the same concentration of primary in both procedures? Same concentration of avidin conjugate?

Why are your protocols so different for the same stain?

Why do you use EDTA in your wash buffer?

Offline apple

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IF is working but IHC doesn't work, is it possible?
« Reply #4 on: December 12, 2005, 04:22:30 AM »
I did all the procedure according to suppliers' suggestion.

Because I used biotin labelled primary ab and avidin labelled FITC. So I didn't use secondary antibody.  My FITC supplier (Vector) suggested me to do a general blocking with 0.1% BSA. My primary ab supplier suggested me to dilute my ab in 0.5% BSA+ 2mM EDTA in PBS. They also suggested me to use 0.5% BSA+ 2mM EDTA in PBS (no Ca+, Mg+) as wash buffer.  I think probably it is because their primary ab's application is for Flow cytometry and FACS. But I just did as they suggested.
I think I should use 1% BSA block not 0.5% BSA. I think BSA conc. for blocking should be higher than ab dilution.

Use methanol fixation is because I read some references and they used methanol and I just want to do a trial to see if there is some difference.


Many thanks for your comments.

Apple

Offline jonnieplumb

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IF is working but IHC doesn't work, is it possible?
« Reply #5 on: December 12, 2005, 05:12:08 AM »
Problem with methanol and CD markers is that methanol can remove these epitopes thus not allowing the Ab to bind. Even when blocking endogenous peroxidase with H2O2 in Methanol can be detrimental and should be done later in the protocol ie after the primary Ab and Biotinylated Ab stage

Offline apple

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jonnieplumb
« Reply #6 on: December 12, 2005, 11:31:39 AM »
Hi jonnieplumb,

Thanks very much. Do you mean apply H2O2 after Biotin labelled primary ab and just before applying Avidin labelled HRP?

Thanks

Apple

Offline jonnieplumb

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IF is working but IHC doesn't work, is it possible?
« Reply #7 on: December 12, 2005, 12:08:35 PM »
Hi Apple
Yep incubate in your biotinylated secondary (eg BRAM) 30 mins RT, then 2x5 mins PBS, then 0.3% H2O2 in methanol 10mins RT, running water 5mins, PBS 5mins, then SABC-Px 30mins RT and so on.
I've been using it for CD4 CD8 and the like in lung tissue.
Good luck
J

Offline richard03

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IF is working but IHC doesn't work, is it possible?
« Reply #8 on: December 12, 2005, 05:27:09 PM »
I have to agree with MaximinaNYC and jonnieplumb that the Methanol may be the main cause for your ICC protocol not working. Read the following tips and it may be helpful.

http://www.ihcworld.com/_technical_tips/crystat_section_ihc.htm

Richard

Offline ImmunoNYC

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IF is working but IHC doesn't work, is it possible?
« Reply #9 on: December 12, 2005, 06:55:18 PM »
Why not just eliminate methanol altogether? It's simply not worth the risk ...  I block endogenous peroxidases in 0.3% H202 in H20 to my satisfaction in 9/10 cases.

I do on occasion use methanol as a fixative but only in cases where I am sure the antigen is maintained.


Quote from: "jonnieplumb"
Hi Apple
Yep incubate in your biotinylated secondary (eg BRAM) 30 mins RT, then 2x5 mins PBS, then 0.3% H2O2 in methanol 10mins RT, running water 5mins, PBS 5mins, then SABC-Px 30mins RT and so on.
I've been using it for CD4 CD8 and the like in lung tissue.
Good luck
J

Offline apple

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IF is working but IHC doesn't work, is it possible?
« Reply #10 on: December 13, 2005, 09:27:15 AM »
Thanks everybody.  Thanks a lot for your suggestion.

Regards

Apple

Offline apple

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IF is working but IHC doesn't work, is it possible?
« Reply #11 on: December 13, 2005, 09:02:08 PM »
Hi,

Just read one reference, for blocking endogenous peroxidase, they used a solution containing sodium azide (1mM), glucose (10mM), and glucose oxidase (1U/mL) for 60 min at 35C.  Why did they use this "strange solution" not H2O2?

Does somebody know this?

Many thanks

Merry Xmas!!!!

Apple

Offline ImmunoNYC

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IF is working but IHC doesn't work, is it possible?
« Reply #12 on: December 13, 2005, 11:39:10 PM »
This is my favorite method for tricky sections/preps. It provides a very slow gentle method to effectively quench all peroxidase activity. It is really only needed if staining tissues of hematopoietic origin - I believe it's the neutrophils that cause the problem. I live for this method for spleen and bone marrow.

For routine IHC, ICC etc I use H202 or alternatively a commercial peroxidase blocker from DAKO as the method you mention takes 1 hour. Time is precious!!  :wink:


Quote from: "apple"
Just read one reference, for blocking endogenous peroxidase, they used a solution containing sodium azide (1mM), glucose (10mM), and glucose oxidase (1U/mL) for 60 min at 35C.  Why did they use this "strange solution" not H2O2?

IF is working but IHC doesn't work, is it possible?
« Reply #12 on: December 13, 2005, 11:39:10 PM »