[Histonet] (no subject)

[Amos Brooks]

Hi Tresa,
     I haven't seen any responses to this question, so I figure I should take a crack at it.
     You are right that using a polymer detection system is so clean that you hardly have much to worry about background/non-specific staining when not using a buffer for wash steps. There is however a good reason to use them anyway.
     To explain this it might help to think of two slinkey springs laying parallel to each other. You need to get them to slide together as perfectly as possible. If they are moving around like slinkeys always do, this will be a difficult thing to accomplish. You will need to get them to sit still so that when you push them together they will just slide nicely into place.
     Taking this analogy to the realm of IHC that we are discussing, the two slinkeys are obviously the antibody and antigen. The wiggling and jiggling that they are doing is due to Van der Walls forces (I probably misspelled that). Now we usually have a few strategies to make these jiggley antibodies behave. Having a good antibody diluent helps a lot, as does having an antibody that has a high affinity and avidity to the antigen. It also helps to have the keep the reaction at a constant pH and tonicity. That is where the wash buffer comes in. It can keep the non-specific staining out when using a avidin-biotin reaction, but it also makes the antigen-antibody reaction more comfortable.
     So, yes you will surely see some decent results with distilled water washes especially with antibodies that are really attracted to each other, but for the ones that need some more coaxing or the ones that you are *really* depending on specificity for quantitation (her2, ER, PR etc), you will really want to make sure you have given these two love bird slinkeys as much chance to hook up a  possible.
     I hate trying to explain something like this without citing a good source for where I got this from. In looking around there isn't much out there explaining the "Why" of many IHC procedures. So much of modern medicine is voodoo just follow the procedure, dont ask questions! This is why I always recommend the Dako IHC manuals. They give such explanations without being so verbose you want to put your eyes out. So for more discussion on this subject check out  the Dako IHC manual 5th edition pg 122 or thereabouts.
     I hope this helps explain why I feel the buffer steps are essential and really shouldn't be replaced by water rinses. It is a really good question though since we are all trying to save a little money and with how much companies are charging for these buffers, it's a good place to look. IHCworld has some great recipes for making them up yourself and they will save you a fortune.

Have a great weekend,
Amos


Message: 1 Date: Thu, 13 Mar 2014 17:16:52 +0000 From: "Goins, Tresa" <TGoins <@t> mt.gov> Subject: [Histonet] IHC without wash buffer To: "histonet <@t> lists.utsouthwestern.edu" <histonet <@t> lists.utsouthwestern.edu> Message-ID: <FA81E534F40DEF44A70F0FBBCD49D2E425D47366 <@t> doaisd5235.state.mt.ads> Content-Type: text/plain; charset="us-ascii"

In a continuing effort to limit the volume of reagent used in each step of a manual IHC, I have tried TBS and TBST on slides with barriers (expensive) and without barriers. The results were not stellar with the barrier slides - the reagent still escapes. We dewax with hot detergent (may be a contributing factor) and the Tween 20 in the TBST definitely alters the hydrophobicity of the barrier, an effect that is not reversed with a water wash.

Consequently, I have resorted to omitting the buffer wash steps and using distilled water only. The slide surface remains "water repellent" and the added IHC reagents form a pool over the tissue sections. The detection method is a polymer-HRP and there is no increase in background staining in the tissue or on the slide surface.

I am assuming that the IHC reagents are prepared in the "optimum" suspension liquid and a buffer is not required. So, I am interested in hearing from anyone why this is a bad idea.

Thanks,

Tresa