Author Topic: Same Host Species Seq. Double Staining... Am I doing this right?  (Read 9317 times)

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Offline NeedSomeHelpPlz

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I'm currently troubleshooting an ICC protocol for the lab I work in. No one in my lab is a master of fluorescence microscopy, so I'm really on my own here and I'm trying to determine whether the results I'm getting on my slides are legitimate or incorrect. I'm performing double stain preps in chamber slides, formulating baseline comparisons between total alpha tubulin, acetylated tubulin, and tyrosinated tubulin levels in PC12 neurons. Now, in referring to other research, I have come up with some expected visual results (relative localizations of acet-tub and tyr-tub) and I've devised a protocol that I would like feedback on. Currently, I have troubleshot a LOT of slides, running a sequential double stain protocol using the same host species primaries. I have manipulated concentrations, stain durations, block steps, etc. in order to obtain the best visual results for hopeful qual- and quantification and ALL of them aren't demonstrating what I had expected, so either my expectations were way off, or my protocol is flawed :/. I just can't seem to figure out where I've gone wrong and I'm having a hard time swallowing the possibility of four wasted weeks due to an incorrect procedure. Please help with any/all suggestions as to what I am doing wrong, or right. Thank you. My list of components and staining protocol is as follows:

Component List:
     Blocks: 1. Jackson Immuno Normal Goat Serum
               2. Jackson Immuno Normal Mouse Serum
     Primaries: 1. Sigma (Mouse) anti-alpha-tub unconj. (IgG1)
                   2. Sigma (Mouse) anti-alpha-tub-acetylated (IgG2b)
                   3. Sigma (Mouse) anti-alpha-tub-tyrosinated (IgG3)
     Secondaries: 1. Molec Probes Goat-anti-mouse Alexa Fluor 555 (IgG)
                       2. Molec Probes Goat-anti-mouse Alexa Fluor 488 (IgG)
     Antifade: Prolong Gold w/DAPI

(this is post-paraformaldehyde fixation, for the sake of simplifying what I would like advice on)

1. 30 min. block with 10% Goat Serum in PBS-Tx, followed by removal of block, but not a wash step.
2. Administration of first primary (either acetyl tub or tyros tub), 1:200 in PBS-Tx. Sit for 1 hour.
3. PBS wash 3x5min
4. Administration of 1:800 (in PBS-Tx) Goat-anti-mouse Alexa Fluor 555. Sit for 1 hour.
5. PBS wash 3x5min
6. 30 min. block with 10% Mouse Serum in PBS-Tx, followed by removal of block, but not a wash step.
7. Administration of second primary (alpha-tub-unconj), 1:200 in PBS-Tx. Sit for 1 hour.
8. PBS wash 3x5min
9. Administration of 1:400 (in PBS-Tx) Goat-anti-mouse Alexa Fluor 488. Sit for 1 hour.
10. PBS wash 3x5min
11. ProLong Gold w/DAPI admin and coverslipping.

Results: while the 555 and 488 show up well and the intensity is comparable, the localization of the respective variants of tubulin is at best unclear... in some cases the slides are resembling f-actin stains or scanning EMs!!! Please, please, please help me. Any sound advice is welcome.

Sincerely,

Frustrated Researcher


Offline gula

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Re: Same Host Species Seq. Double Staining... Am I doing this right?
« Reply #1 on: June 29, 2011, 10:57:18 AM »
My question is: why do you think, that the second-applied secondary anti-mouse-antibody does not bind to the first mouse-primary antibody?

The staining is on human tissue?
gula

Offline NeedSomeHelpPlz

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Re: Same Host Species Seq. Double Staining... Am I doing this right?
« Reply #2 on: June 29, 2011, 11:13:14 AM »
Gula,

I would hypothesize that the mouse block (2nd block step) would take care of any available binding sites left from the first set of primary/secondary antibodies? I honestly don't know... as I'd said, this is new to me and sadly I'm the guinea pig on this one and honestly what you've suggested is almost precisely what my slides are looking like under the scope... they are looking like the 2nd antibody set isn't binding what it should (or more than it should) :( What would you recommend then? How could I do this more effectively, using what I have available, to make sure that the 2nd secondary isn't in fact binding to the 1st primary sites? Is this even possible for me to pull off correctly?

Sincerely,

Now even more frustrated researcher

p.s. the cell line is Rat (PC12) derived from a neuroendocrine tumor of the adrenal medulla

Offline MT Scientist

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Re: Same Host Species Seq. Double Staining... Am I doing this right?
« Reply #3 on: June 29, 2011, 04:50:13 PM »
There is where your problem probably lies.  Try WITHOUT the mouse serum block (should be normal goat serum or even PBS-Tween with or without BSA).  Abs from the mouse serum could be binding to the goat anti-mouse (or to the tissue) and allowing for the binding of the 2nd goat anti-mouse resulting in the non-specific staining.
« Last Edit: June 29, 2011, 04:52:08 PM by MT Scientist »

Offline NeedSomeHelpPlz

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Re: Same Host Species Seq. Double Staining... Am I doing this right?
« Reply #4 on: June 30, 2011, 10:26:13 AM »
MT,

Do you mean without a 2nd block step altogether, or replacing the mouse serum with goat serum (same as first block)? Also, we use PBS-Triton-X and are you suggesting that I could just perform a Tx wash instead of the second block, prior to administering the 2nd primary? I'm eager to test out your point, I just want to make sure I'm understanding it correctly.

Thanks

Offline MT Scientist

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Re: Same Host Species Seq. Double Staining... Am I doing this right?
« Reply #5 on: June 30, 2011, 11:58:39 AM »
Replace the mouse serum block with a blocking step using goat serum (or PBS-T with goat serum).  Not blocking at all could cause other problems.  Make sure you have a good wash step (PBS-T) after your first Alexafluor as they can be "sticky".  

You could also try Tween-20 instead of Triton-X.

Titrate your primary and secondary antibodies to effect.  Too much or too little of the primary and/or secondary can cause background issues.

Look at this staining procedure as two separate sequential IF stainings.  Shouldn't matter that you are using two mouse primaries IF you get the blocking right....
« Last Edit: June 30, 2011, 12:03:12 PM by MT Scientist »

Offline gula

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Re: Same Host Species Seq. Double Staining... Am I doing this right?
« Reply #6 on: June 30, 2011, 12:12:05 PM »
You can also try a heat-denaturation step after the first secondary. I think, someone reported in this forum, that it worked.
Heating in Retrievalbuffer, 75C, 5-10 min. This should denaturate your first primary and you get a "brand new" slide for the second part of your doublestain. The question is, if the fluorochrom will survive.
This methods works well with Peroxidase+DAB / Alkalic Phosphatase+Red on tissueslides.
Your cells are fixed, so they could withstand the heat.

Gula

Offline MT Scientist

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Re: Same Host Species Seq. Double Staining... Am I doing this right?
« Reply #7 on: June 30, 2011, 12:38:36 PM »
You can also try a heat-denaturation step after the first secondary. I think, someone reported in this forum, that it worked.
Heating in Retrievalbuffer, 75C, 5-10 min. This should denaturate your first primary and you get a "brand new" slide for the second part of your doublestain. The question is, if the fluorochrom will survive.
This methods works well with Peroxidase+DAB / Alkalic Phosphatase+Red on tissueslides.
Your cells are fixed, so they could withstand the heat.

Gula

Don't know if it would, but it seems a possibility that denaturing the first primary (with the fluorescent secondary attached) could lead to loss of signal.  I can't say for sure.

This could be used to denature endogenous antibodies prior to doing some immunos (e.g. denature endogenous mouse antibodies prior to using a mouse primary and an anti-mouse secondary)....

Offline gula

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Re: Same Host Species Seq. Double Staining... Am I doing this right?
« Reply #8 on: June 30, 2011, 12:56:27 PM »
With fixed tissue or cells endogenous antibodies are also fixed/denatured. IHC-antibodies are native and therefore are not recognised by the secondary, if you denaturate them by heat. Perhaps there is also a wash-away effect.
With DAB-staining the system works well, because DAB binds very sticky to the tissue.
In this special protocol the fluorochrom is bound through the antibodies to the cell-proteins. Antibody gone - fluorochrom gone? Perhaps a ligation step with formaldehyde will bind the fluorochrom to the proteins and helps to survive.

This considerations are only theoretically - never proofed it.

gula

Offline NeedSomeHelpPlz

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Re: Same Host Species Seq. Double Staining... Am I doing this right?
« Reply #9 on: July 03, 2011, 06:55:45 PM »
Look at this staining procedure as two separate sequential IF stainings.  Shouldn't matter that you are using two mouse primaries IF you get the blocking right....

Great suggestion, if it works for the first set, maybe it should work for the second as well. I'm psyched to test it out and crossing my fingers. Thanks for the insight MT and Gula, I'll post once I get some results from this next batch of troubleshooting.

Hopeful Researcher


Re: Same Host Species Seq. Double Staining... Am I doing this right?
« Reply #9 on: July 03, 2011, 06:55:45 PM »