1- I read sometimes people added blocking solution after primary Ab and after that added secondary Ab, while, other people normally do before primary Ab and prior that added secondary Ab.
- My tissue is chicken Duodenum
Is there any way to know what time should add blocking reagents?
2- Some papers mention different dilutions in primary and secondary antibodies. How can I be sure for primary which dilution and for secondary which dilution could be work? (Because, till now I've used same dilution (e.x 1/100 or 1/1000) for both primary and secondary antibodies)
In the other word, Is there any way to detect whether I have to dilute primary antibody more or secondary antibody?
3- Is that ok while I am working on chicken Duodenum , my secondary antibody could be Goat Anti-Chicken IgY???
My Secondary Ab:
Goat pAb to chicken IgY (from abcam)
4- My target antigens are intracellular, and I need to use a permeabilisation step. may I know in which step I have to do permeabilisation ? Normally after antigen retrieval and blocking step I do permeabilisation with Triton-x 100 for 10 minutes. Is that correct ?
Till now I did not succeed to detect virus in my tissue, while I detected in ICC(But I needed to detect in tissue as well):
Shall I use longer permeabilisation ?
5- I am using 1% Normal Goat Serum as a blocking step before primary Ab step, shall I increase it to 5%?
6- While I am using Goat Anti-Mouse IgG3 (Secondary Antibody) for my targeted cells, is it ok in a same time also I use Goat pAb to chicken IgY (Secondary Antibody) for detection of my targeted virus in my chicken tissue?
7- For washing step, I read different concentration of Tween 20 such as 0.5 %, 0.1%, 0.2% and 0.05%, which one should I take it? It is confusing....
8- Some references says use 5% Normal Goat Serum for blocking step and also 5% Normal Goat Serum for Ab dilutions. while, others says 5% Normal Goat Serum for blocking step and 1% Normal Goat Serum for Ab dilutions. which one is correct?
I appreciated in advance for your help
So in order,
1) I block before addition of primary antibody and that's it. At this point if you block for a long enough time you should have covered all the sites where your antibody could have bound non-specfically. Not saying that its absolute but generally its your primary binding to non-specific sites that gives you background. Also for anti-chicken antibodies, there are specific blocks for these that you can get. The few chicken antibodies I have used have quite high background unless you use these particular anti-chicken block.
2) It depends on the primary and secondary. The primary dilution will have to be determined through testing it yourself unfortunately or using dilutions that people have used this particular antibody at or what the company recommends it at. However, the best way is to start with lower dilution (1:100) and then do further dilutions later once you know that works, the lower the dilution also the less chance for background staining. For the secondary, I have never used anything but the alexa fluro's and those don't give much or any background for the most part. I use those at 1:400 always. But if using others, you will probably need to try it out to see, you might start at 1:500 for the secondary and then dilute further if you see any non-specific staining in a secondary only control slide.
3)seems fine, what is your primary?
4) You should permeablise while you do your first blocking step. The block should contain your block (BSA, serum or antichicken block) plus triton-X 0.1%. Block for at least an hour. And you can do all your washes afterwards with this block.
5) I don't usually use serum as a block, for whatever reason I find serum to be noisier than BSA as a block. But since you have chicken tissue you might want to try the stuff they sell of chicken specifically. But if not, I use 2% BSA for regular mouse tissue, but if that is still noisy you can go up 5% BSA or serum. But block for an hour minimum, that should help.
6) you are looking for 2 things? I am confused, you are using chicken tissue but where does the mouse antibody come in, the goat anti-mouse. Where is the mouse tissue?
7) I would just wash with your block that contains triton-X. Tween is just a gentler surfactant but I've never found a difference so unless there is a specific reason to do so, just use the same wash as your block, saves time and you don't have to make up several different washes.
8) There is no real correct answer, its whatever works for you. I always put the primary in the same blocking solution I use to block. So the block first 1hour (2%BSA, PBS + 0.1% triton-X or tween-20 if you want to just use tween). Then I add primary to the block and add to the tissue. The primary can go for 1hour room temperature or O/N 4C. After primary, rinse 3x block, wash 3x 10 minutes blocking solution (but the wash can be increased up to 30 min each time if you are worried, i increase washing time depending on how thick the tissue is, for whole embryos, I wash 30 minutes each time but for cells or sections, 10 minutes each. After washing, add secondary in blocking solution 1 hour room temp. I never put secondary on overnight, that is too long and you will get non-specific staining. after secondary rinse and wash, add dapi, rinse and mount.
All of this is subject to tweaking for your particular tissue and antibody and unfortunately it depends on how fussy your antibodies are and your particular tissue but this is a basis to start. Also, the reason you see all these different protocols in papers is this is what works for them or whatever their lab protocol has always been, not because its necessarily the best protocol.
So in order,
1) I block before addition of primary antibody and that's it. At this point if you block for a long enough time you should have covered all the sites where your antibody could have bound non-specfically. Not saying that its absolute but generally its your primary binding to non-specific sites that gives you background. Also for anti-chicken antibodies, there are specific blocks for these that you can get. The few chicken antibodies I have used have quite high background unless you use these particular anti-chicken block.
2) It depends on the primary and secondary. The primary dilution will have to be determined through testing it yourself unfortunately or using dilutions that people have used this particular antibody at or what the company recommends it at. However, the best way is to start with lower dilution (1:100) and then do further dilutions later once you know that works, the lower the dilution also the less chance for background staining. For the secondary, I have never used anything but the alexa fluro's and those don't give much or any background for the most part. I use those at 1:400 always. But if using others, you will probably need to try it out to see, you might start at 1:500 for the secondary and then dilute further if you see any non-specific staining in a secondary only control slide.
3)seems fine, what is your primary?
4) You should permeablise while you do your first blocking step. The block should contain your block (BSA, serum or antichicken block) plus triton-X 0.1%. Block for at least an hour. And you can do all your washes afterwards with this block.
5) I don't usually use serum as a block, for whatever reason I find serum to be noisier than BSA as a block. But since you have chicken tissue you might want to try the stuff they sell of chicken specifically. But if not, I use 2% BSA for regular mouse tissue, but if that is still noisy you can go up 5% BSA or serum. But block for an hour minimum, that should help.
6) you are looking for 2 things? I am confused, you are using chicken tissue but where does the mouse antibody come in, the goat anti-mouse. Where is the mouse tissue?
7) I would just wash with your block that contains triton-X. Tween is just a gentler surfactant but I've never found a difference so unless there is a specific reason to do so, just use the same wash as your block, saves time and you don't have to make up several different washes.
8) There is no real correct answer, its whatever works for you. I always put the primary in the same blocking solution I use to block. So the block first 1hour (2%BSA, PBS + 0.1% triton-X or tween-20 if you want to just use tween). Then I add primary to the block and add to the tissue. The primary can go for 1hour room temperature or O/N 4C. After primary, rinse 3x block, wash 3x 10 minutes blocking solution (but the wash can be increased up to 30 min each time if you are worried, i increase washing time depending on how thick the tissue is, for whole embryos, I wash 30 minutes each time but for cells or sections, 10 minutes each. After washing, add secondary in blocking solution 1 hour room temp. I never put secondary on overnight, that is too long and you will get non-specific staining. after secondary rinse and wash, add dapi, rinse and mount.
All of this is subject to tweaking for your particular tissue and antibody and unfortunately it depends on how fussy your antibodies are and your particular tissue but this is a basis to start. Also, the reason you see all these different protocols in papers is this is what works for them or whatever their lab protocol has always been, not because its necessarily the best protocol.
Dear Victoria, first of all, thank you for your concern and replying.
1- You mention that there are specific blocks for anti-chicken antibodies. May I know more about it if you have any information?
4- If I get you correctly, you mean e.x I have to use in a same time blocking and permeablise together (in this case 5% Normal goat Serum + 0.1 % Triton X-100) for one hour?
- Also you mention that I can use this mixed blocking afterward as a washing also? The problem is I have just enough Normal Goat serum to prepare as blocking and do not have enough for washing step…. what can I do afterward? Can I proceed for washing with PBST? Or PBS + BSA??? And if yes, which concentration do you suggest (either for PBST or PBS+BSA)?
6- Yes, I do. My targets are Cells and Virus. My primary antibody for Cell is from Mouse and its secondary also is Goat Anti Mouse and I succeed to detect my targeted cells in chicken tissue and I don’t have problem about that. My Virus primary antibody is Chicken pAb which its secondary antibody is Goat pAb to Chicken IgY which I haven’t found yet the virus in my targeted tissue (I have to mention that I tested Virus antibody separated and not mix with my targeted cell antibody)
>>> Other question:
If I mix blocking and permeablise together as you mention before, after that I have to proceed for primary antibody. I want to know after this blocking and permeablise step, shall I wash and then do staining or straight I do primary staining? (Because this time Triton-X also is mixed with blocking reagent, I doubt shall I wash or do not)
- also I found this protocol (attached file) which after primary and secondary antibody, they washed with PBS+0.2% Triton X and after that washed again with PBS. Do you have any idea about it?
Hi,
1) its this stuff called BlokHen from Aves lab
4) yes use the block and permeablise step together, so your block will have both 0.1% triton or tween and your goat serum, but if you don't have enough serum you can use BSA instead. If you just have enough goat serum from blocking, then you can subsequently wash with just pbs + triton with or without BSA. I've never mixed different blocks before though technically it shouldn't matter but if you have BSA I would just use BSA at 2%, its cheaper and I prefer it. Though if you get the chicken block I forgot what happens after you block with it, but it will come with directions.
6) i misread this question. The combination should be fine.
After blocking and permeablization go straight to staining, no need to wash. I think in the paper that is what works for them and is what they teach eveyone in the lab. There is no good reason to wash again with PBS.
Good luck
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