What is the role of NaOH and SDS in immunohistochemistry protocols?

[Moonbear]

Okay, this one has me baffled. I'm hoping someone else here has some ideas (DocToxyn, perhaps?). I'm attempting an IHC protocol based on a previously published protocol. The published protocol seems a bit odd, but trying to skip the "oddities" didn't result in any immunoreactive cells showing up (I ran a fairly extensive dilution series with the primary antibody). This is confounded a bit by the fact that I'm trying this in a different species than the original publication, so don't actually know for certain that I should see expression. (Yeah, yeah, I'm going to have to resort to just following their protocol exactly, odd things and all, but figured I'd give it a first stab with a more standard protocol just in case something simple worked.)

Anyway, I'm trying to understand the purpose of the added steps since I've never seen a protocol include them before, so don't know what they do to help. If I understand the theory, I might be able to troubleshoot more easily. Besides, if I get it working, I then need to teach this to the folks in the lab I'm doing it for, so I want to know what these added steps are doing so I can explain it as something other than, "because the protocol said so, and it doesn't work without it."

One thing that was added was NaOH in the peroxide blocking step...the article says 1% NaOH was added (but they don't say what the pH of their buffer started out as...I'll have to call and ask, but for the moment, I'm assuming it was the usual pH 7.3-7.4). Is this some sort of antigen retrieval method I've never heard of? The alternative explanation is that for some reason, the authors used PBS when they really needed TBS and the higher pH range you get with that.

I'm trying to decide if my next attempt should be with PBS at a higher pH, or just switch to trying TBS at pH 8.0. If nobody here has any ideas on that, I'll probably just try both next time around.

The other thing they added was SDS. I can't really figure out why you'd want to use SDS in an IHC protocol. They used that in addition to triton-X100. Would that suggest that the immunoreactive portion of the protein is inaccessible to the antibody in its normally folded state? Is there some other reason you'd use SDS in an IHC protocol? Or is this a case of some student confusing the Western protocol with the IHC protocol and nobody caught it until they were getting ready to publish? :uhh:

My best overall guess is that the antibody is raised against a portion of the protein that is not very accessible, and you need to beat the cells into submission to get access to it.

 

[Ouabache]

Where are all the immunohistochemists when you need one?

Since you really want to learn the underlying chemistry involved,
I wonder if you crossreferenced this query under chemistry, you may
get some good replies.

 

[Monique]

From: http://www.ihcworld.com/_protocols/immunofluorescence/cellular_structure.htm

"Following fixation, I have found that permeabilization with 0.5% SDS for 10 minutes consistently yields the best results in terms of uniformity of staining as well as decreased background fluorescence."

I don't know about the NaOH, they used paraformadehyde by any chance? NaOH helps it dissolve, but that really is a stretch.

 

[Moonbear]

From: http://www.ihcworld.com/_protocols/immunofluorescence/cellular_structure.htm

"Following fixation, I have found that permeabilization with 0.5% SDS for 10 minutes consistently yields the best results in terms of uniformity of staining as well as decreased background fluorescence."

Interesting. I was talking with someone else, and she said she'd tried SDS in a protocol once, and all it seemed to do was increase background. :uhh: Well, at least it means someone else has done it with success, so I'll add that in and see what happens.

I don't know about the NaOH, they used paraformadehyde by any chance? NaOH helps it dissolve, but that really is a stretch.

That's for making the paraformaldehyde, not in the IHC. I wish they had indicated the pH of the starting or ending solution. For all I know, they could have had someone making all their PBS too acidic and all the NaOH did was put it back in the physiological range.

 

[DocToxyn]

I don't have a huge amount of experience with IHC, but I've never seen NaOH included in that fashion in any methods I've either performed or read about. So the NaOH was included in a hydrogen peroxide-based step to eliminate endogenous peroxidase activity? Yeah, it could have simply been a buffer pH thing, or perhaps they have found that the blocking step is more effective with the NaOH included (again probably a pH issue)?

As far as the SDS, perhaps they just went on the idea that two detergents are better than one? I use both sodium deoxycholate and NP-40 when I do whole mount fetus staining for beta-galactosidase reporter gene signal. I've seen other methods that don't use any detergents or others than use either one or different ones. I hate to say it but, like you, it's another case of well this is what the method calls for so that's why I did it...although the reason for the detergents is well known to aid in penetration of the reagents. Plus, as Monique states, other methods use a variety of detergents- Tween-XX, SDS, triton, etc so maybe they just tried what was on hand and it worked.

Did you try it "their way" yet?

 

[ladsh]

I have a protocol that includes both those steps. I have never found a good explanation (actually I've never really had any explanation as to why these steps are done). I originally started to use this protocol because the few other articles that had used the antibody had all done these steps. I would like to find that original person and see what they were thinking at the time! I always followed this 'crazy' protocol and my staining was beautiful everytime. I stopped asking why and just decide it was something i would never know. I wanted to try the protocol without these steps but I never got around to it since it worked so well. For the first incubation with hydrogen peroxide and NaOH I never used TSB or PBS, I just used water. I understand why you want to know why these steps are used but if the staining is working why question it so much (this is the one thing in my science career that I have had to just accept and stop questioning!). If you do ever find out why, please make sure you post the answer. Sorry I couldn't include any answers. You never did say if your staining was working (or did i just miss that part)

 

[Moonbear]

I haven't had time to get back to it, but clarified part of the mystery of why my staining didn't work on the first try...the technician ordered the wrong antibody. Apparently nobody has ever gotten the one that was ordered to work, but she forgets this and keeps reordering it whenever someone asks her to order the antibody for this protein. I have the right one to try now, but just have not had time to troubleshoot it. I'll provide an update when I get around to it if I find anything new, or find that the "weird stuff" in the protocol is totally unnecessary.

 

[Moonbear]

Okay, I promised I'd get back to this thread when I had time to play more with this protocol. I got it working (or think I have), and sure enough, it really does require both the NaOH and SDS. It's not just a pH issue, because my attempt at using tris buffer at pH 8 didn't do a thing. I'm running controls this week along with a few more variations to see if it can be refined any further. It appears it is some sort of antigen retrieval method, but the background is amazingly cleaner (as in there isn't any background...unless it's all background ...that's what the controls are for) than when I've used other antigen retrieval methods. I finally got my paws on the spec sheets for the antibody too, and those recommend boiling with citrate buffer as an antigen retrieval method. I'm going to try that too, just for comparison. It could be that this antibody really is that clean that I get so little background, but I want to know how a more familiar antigen retrieval method compares. If this method really has much cleaner background, I might have to give it a try for a few other stubborn proteins that require antigen retrieval to detect but then are plagued with background problems.

And, of course I still need to find out if it's compatible with the other stuff I want to be able to double label with this protein.

Oh, and in this protocol, the peroxide step is combined with the NaOH step, which explains why it can be done in just water. The NaOH concentration ends up being 0.25M (why they had to express it as a percent, I don't know), so the osmolarity of the solution isn't so far off from the buffer solutions.

 

[Moonbear]

Just another update, if anyone else cares or would benefit from it. You definitely need BOTH the SDS and NaOH in the protocol, though the NaOH is the more important of the two (absolutely no staining if I left out the NaOH step, but some with SDS, though nowhere near optimal). I tried this protocol with another antibody that I have worked with a lot for detecting a cytoplasmic peptide (GnRH), to see if it's 1) going to be compatible in any double-labeling protocols, and 2) what it does for cytoplasmic as opposed to nuclear peptides/proteins. The results...gorgeous! I was able to dilute out the antibody much further (in a quick test, it was still going strong at half the concentration I usually use). And in my primary antibody omission controls...absolutely clean!

If anyone else thinks they would benefit from this method, send me a PM and I can get you the detailed protocol, and the reference I got it from.