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Post subject: When to use Antigen Retrieval Solutions?
Greetings once again, to all my favorite histologists and immunohistochemists...
Since it has been about one-week since my last post, I will try and update everyone on my project...
I have completed the required double-labeling / immunofluorescent studies of INGAP vs. Glucagon in the mouse and hamster; we have now moved on to commercial slides of rat and monkey. Oooh, how exciting, no?
I still have to contend with the RBC autofluorescence, but hey, what can a girl do...
However, today's post is in reference to Antigen Retrieval solutions...
When I first began working here, we attempted to use an Antigen Retrieval solution on our INGAP, to try and enhance our overall signal (since it was so poor to begin with); however, and looking back at the slides now (three, almost four months later), I have come to realize that there was NO difference in the INGAP signal with the Antigen Retrieval, than the one I see now (after four months of desperation, and one protocol change)...
Yet, I have read various articles / posts of individuals that have used the Ag retrieval solution with wonderful results...
Could this be due to the INGAP antibody? Is there the potential that certain antibodies will just NOT react to Ag retrieval solutions?
Alas, I shall wait (by my computer) for an answer...LOL...or at least, until my timer goes off.
With all my love,
-MC, the perplexed
Mon Feb 19, 2007 8:47 pm
Carl
Rank: Member
Joined: Oct 11, 2004
Posts: 1871
Post subject: Lol....
We are all favourites of MC now....eat your heart out Ole
However, MC, as it has been a full week...remind me what tissue prep you are using? ( saves me having to dig into archives...sigh, such an effort!)
Re your Q: sure....there is never any guarantee that any Ab will work specifically in any particular situation. I have many Abs that work in WBlots but will never work in pwax/frozen sections....and alternative combinations eg: great in pwax/frozen sections but not in W .....
I have many Abs that also react non-specifically.........but give damn fine staining ! I have to be very carefull.....
It's a minefield ...tread carefully. Blows one's head , forget the leg!
Sure, the established Abs are fine...they have taken a few years to be validated. (like L26 clone for Bcells...but still the occassional paper throws up "weird" staining....it's an ongoing process)
IMHO......
Carl The Careful
Peer review, peer review;-)
Mon Feb 19, 2007 9:12 pm
MC
Rank: Member
Joined: Feb 07, 2007
Posts: 11
Post subject:
My dear, sweet, and all-knowing Carlitos...
Of course I am fond of all of you...what would I do without my Carlitos and Ole (and don't forget Hogne too)!?!
With respect to my tissue prep...After I harvest the pancreas(es), I fix in formalin, and then ship them out to our pathology departmet for paraffin embedding.
Does that answer your question? Or, do you need something more specific???
*Still waiting at the computer...LOL
-MC
Mon Feb 19, 2007 9:23 pm
ole
Rank: Member
Joined: Oct 11, 2004
Posts: 853
Post subject:
Hello MC
As i have no experience with your ab (INGAP) iys difficult to know what treatment will work best.
But for the large majority results will improve on FFPET using HIER, for many/(most) antibodies it is essential using HIER.
As far as possible i run diffenrent tests on tissues containing both +/- controlles, also if i know of such im using tissues where the anitgen is expressed in a high and low amount.
On these tissues i use;
1. No pretreatment
2. Enzymetreatment
3. Different HIER techniques, different buffers and different temperatures.
If none of these produce the staining i want i try other enzymes, other retrieval buffers, acids etc or combinations.
And i try two sets of different detectionsystems, one giving clean staining and one who usually produce strong staining - a sensitive detection system. If none of this works i order a new ab .
It all empirical.
And even tho it seems to work fine with a given protocol one might need to try it on tissues fixed/processed different. Some protocols (often dependent on ag of interest) will produce very different staining on different processed/fixed tissue. So a protocol might be optimal at first try, others might take weeks and a long range of optimising to get good.
Some ab are crap and seems impossible to get good/optimal.
Of the more or less 300 ab i use regularly i think about;
1-2% works best without antigenretrieval
3-5% using enzymetreatment
90% using different techniques of HIER and retrievalbuffers.
1-2% combination HIER and enzymeretrieval
1-2% Acids or such.
For some of these abs, the staining gets optimal only by using one spesific protocol.
Ole
Mon Feb 19, 2007 9:24 pm
MC
Rank: Member
Joined: Feb 07, 2007
Posts: 11
Post subject:
Hola Ole!
Great to hear from you...
As I mentioned in my very first post, I am a VERY green immunohistochemist - basically, think of me as the VERY bottom rung of a ladder...
Thus, I am slightly lost with your post...to speak in all honesty, I am staring at the computer screen with a blank stare...
What exactly is FFPET and HIER? *hangs head in shame...
Also, the Ag solution that we are using is commercially bought from BioGenex, and is nothing more than a citrate buffer...
Any help or information that you can give me, or direct me to, would be appreciated!
Thanks again, Ole!
(I just love saying your nickname! It reminds me of home...*sighs...)
-MC
Mon Feb 19, 2007 9:34 pm
Carl
Rank: Member
Joined: Oct 11, 2004
Posts: 1871
Post subject:
Quote:
I have come to realize that there was NO difference in the INGAP signal with the Antigen Retrieval, than the one I see now (after four months of desperation, and one protocol change)...
I think I have that Korrektly ......Or not....
But....do you have a "specific" signal??? Or are both irrelevant???
As skoolkids, some boys would ask you to show your "knickers"...I ask you to show me a pic to demonstrate what you mean
My apologies for any offence that I may cause...sincerely unintentional
Ha.....
As I stated before...no guarantee of any Ab reagent working in pwax
( thanks for details)
Carl The Safe geezer....
Mon Feb 19, 2007 9:34 pm
Yoda
Rank: Member
Joined: Mar 18, 2005
Posts: 94
Post subject: Re: When to use Antigen Retrieval Solutions?
MC wrote:
Greetings once again, to all my favorite histologists and immunohistochemists...
Since it has been about one-week since my last post, I will try and update everyone on my project...
I have completed the required double-labeling / immunofluorescent studies of INGAP vs. Glucagon in the mouse and hamster; we have now moved on to commercial slides of rat and monkey. Oooh, how exciting, no?
I still have to contend with the RBC autofluorescence, but hey, what can a girl do...
However, today's post is in reference to Antigen Retrieval solutions...
When I first began working here, we attempted to use an Antigen Retrieval solution on our INGAP, to try and enhance our overall signal (since it was so poor to begin with); however, and looking back at the slides now (three, almost four months later), I have come to realize that there was NO difference in the INGAP signal with the Antigen Retrieval, than the one I see now (after four months of desperation, and one protocol change)...
Yet, I have read various articles / posts of individuals that have used the Ag retrieval solution with wonderful results...
Could this be due to the INGAP antibody? Is there the potential that certain antibodies will just NOT react to Ag retrieval solutions?
Alas, I shall wait (by my computer) for an answer...LOL...or at least, until my timer goes off.
With all my love,
-MC, the perplexed
Hi MC,
Antigen retrieval could be a lot more complicated than just boiling the tissue in a buffered solution. If I recall well, the first publication the author's try to overcome the overfixation of the tissue to gain a better signal with their commercial antibodies, most antibodies tested improve the signal when treating with antigen retrieval ( or HIER) procedures. These laboratories aim to use these procedures also because they work as reference laboratories which held tissue in 10%NBF and later wanted to performed IHC. On the other side you can also explorer which is the best fixative for the markers you want to stain and fixation time. It's important to established these parameters to builts a better understanding of your stains. Over twenty years ago, the commercial polyclonal antibody for S-100 was not reccomended for frozen tissue (frozen tissue commonly fixed in acetone), I remember that a colleague from Memorial Sloan Kettering Cancer Ctr. She explorered a different fixation on frozen skin an accomplished the stains with great accuracy, all Langehans cells were positive and beautiful [without background], which commonly happens when fixed in acetone.
In recent publication the use of citraconic anhydride is mentioned to be superior to citric or citrate buffers. I'm also an explorer of this vast universe of antigens and procedures. MC, there is a lot to explorer in the IHC universe. The trip is sometimes short of it could take some light-years.
May the force be with you,
Yoda
Mon Feb 19, 2007 9:35 pm
Carl
Rank: Member
Joined: Oct 11, 2004
Posts: 1871
Post subject: Hey! Yoda...
I am Humbled by your post!
Mon Feb 19, 2007 9:37 pm
MC
Rank: Member
Joined: Feb 07, 2007
Posts: 11
Post subject:
Carlitos...
Thanks again...and you don't have to worry about me...I'm Spanish...we never take offense to anything...and plus, I may be "green" to this profession, but I am old in years...LOL!
I do have a specific signal, but what I am not seeing is any additional vibrancy, or the signal becoming much easier to see.
With respect to the pictures, I will in fact attach some, but it will probably be tomorrow morning...I have been in lab since 7:00 am, and I must get ready to go home for the evening.
So, thanks once again, my sweet and safe geezer...and I look forward to "speaking" with you tomorrow!
*Blows kisses for Carl and Ole!
-MC
Mon Feb 19, 2007 9:39 pm
Carl
Rank: Member
Joined: Oct 11, 2004
Posts: 1871
Post subject: Bon Nuit, Mc.....
xxxxx
Mon Feb 19, 2007 9:42 pm
ole
Rank: Member
Joined: Oct 11, 2004
Posts: 853
Post subject:
Dont write so quickly i use so much time squeezing my brain with this overvocabulary language
I use the one-finger-eagle-method, getting a overview and quickly hit eg the letter D with my right forefinger just partly true.
FFPET; Fomaline fixed paraffine embedded tissue, its when combining both aldehydfixation and parafineembedding HIER have its major benefits.
HIER; Heat induced epitopal retrieval. Just heat and a solution/buffer.
Just citratebuffer might not be good enough. I sometimes overdo it, but try. TE, citrat, citrasonic, TRS pH6,1 (purchased), Tris pH 10, and even Tris/HCl buffer when all hope seems lost.
Mon Feb 19, 2007 9:53 pm
ole
Rank: Member
Joined: Oct 11, 2004
Posts: 853
Post subject:
Ole is one of the most common names up here. Guess its derivated from Olav, a common king name up here. If i remember correct one of our Olav`es almost concurred a nation just across the sea (Carl )
I was at "party" not long ago and approx 50% of the men had Ole and something as a first name. Many "shouted" Ole but there where no bull
Ole
Apologies should be Harald the almost conqueror.
Mon Feb 19, 2007 10:06 pm
MC
Rank: Member
Joined: Feb 07, 2007
Posts: 11
Post subject: *Sighs...
Unfortunately, it has been a VERY long day at work, here in the lab...
For this reason, I have not had the chance to upload the images to share...Sorry!
Plus, I now have to prepare for a presentation tomorrow (with a guest scientist), and I was only told 7 minutes ago!
*Slams head on lab bench...
I will try and place the pictures on here tomorrow, Carlitos...
Until then...Have a great night, Carl and Ole! Kisses!
-MC
Tue Feb 20, 2007 8:39 pm
Carl
Rank: Member
Joined: Oct 11, 2004
Posts: 1871
Post subject: No rush...manana, manana
Good luck with your talk!
Get your red cape out, wave it at the audience who are listening to your talk ( some may be asleep ....and shout "Ole!" He will thus appear as a Caped Crusader and wake them up.
Carlos
PS: Anyone: can I put that wavy line above "n" in manana on a UK keyboard?
Tue Feb 20, 2007 8:50 pm
MC
Rank: Member
Joined: Feb 07, 2007
Posts: 11
Post subject: *Laughs...
Thanks Carlitos...
I needed to laugh!
I can only imagine Ole dressed up as the Caped Crusader, but of course, you would have to be there too...as his trusty side-kick "Histo Man!"
LOL...
Also, if anyone does figure out how to put the ~ above the letter "n," let me know too! Do you know how difficult it is to type in Spanish without it (and without changing the computer's language)!
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Greetings once again, to all my favorite histologists and immunohistochemists...
