[Histonet] Long reply on problems with hydrogen peroxide blocking

[Gayle Callis]

Hey Amos, 

 

I will be nice!   It is true peroxidase blocking can be a pain.  However, after looking at the PeroxAbolish SDS from Biocare, I did not SEE any of the chemicals involved in this block  so I am skeptical this is the Glucose oxidase method.  Big Ho Hum!   PeroxAbolish could be similar to a KPL peroxidase block from years past but not sure if  KPL still sells it.  KPL block was not an enzyme mixture and had to be used carefully on frozen sections.   Also, Biocare said nothing about blocking pseudoperoxidases but neither did Vector in their method although the latter did provide the reference which I encourage people to read.   The glucose oxidase method was developed for delicate, minimally (acetone) fixed frozen sections and really does work without H2O2 chewing a delicate FS off the slide or damaging morphology.    The bonus:  glucose oxidase method works on FFPE tissues particularly when an  in house H2O2 method fails to remove persistent background caused by peroxidase or pseudoperoxidses.   It could be that Patrick is actually getting rid of peroxidase but NOT pseudoperoxidases with his H2O2 method?   Glucose oxidase is a good old fashion enzyme/substrate chemical reaction:  glucose oxidase +   β D (+) glucose = production of slow steady rate of hydrogen peroxide to remove peroxidase/pseudoperoxidases in tissues and cells.   It  gave us the cleanest background ever for murine CDmarker/HRP- IHC/solvent fixed frozen sections using a DAB enhancer.  We never could  get rid of endogenous peroxidase background with a commercial  low concentration H2O2 peroxidase block.   This is  another long story.  

 

There is no mystery about the protocol and Vector has this on their website (see below)   

 

CAVEAT:   the β D (+) Glucose, 97% pure can be hard to find but here is the source.   MP Biomedicals , beta-D-Glucose Catalog Number: 100953.  Sigma discontinued this glucose some years ago (heavy sigh!).   I will be happy to provide the original publication for this method under separate email.   



Glucose Oxidase Peroxidase Block (GLUOX),  *Jasani et al: 

 

Working Solution

            β D (+) Glucose, 97% pure (MP Biomedicals Cat. No. 100953)        0.180 g  (180 mg)  

            Glucose oxidase (Sigma G 6641)                                            0.005 g  (5 mg) 

            Sodium azide                                                                           0.0065g  (6.5 mg)

            Dulbeccos PBS                                                                                     50 ml

            Do not preheat working solution.  

 

Protocol:

1.         Immerse sections in working glucose oxidase solution 30 min - 1 hr (per cited reference).  Use a 37°C water bath for even heating.  

 2.        After incubation, rinse sections 3X in DPBS, 5 min per change then proceed to IHC.

            

Results:  Clean background without endogenous peroxidase or pseudoperoxidases in minimally fixed (acetone) FS or FFPE. 

 

NOTES:  A.   Glucose oxidase can be weighed into screw top micro-centrifuge tube and stored in a freezer.  Pay attention to the expiration date and storage conditions.  This way, one can make up a  ready to use stock buffer in large quantity i.e. 2 liters or more.  We called this "GLUOX Buffer" due to sodium        azide content.  Weighing out chemicals each time was painful to say the least.   A stock buffer permitted quick preparation for a desired working solution volume  e.g. 50 ml or more.  To prepare desired quantity of working soluton:  pipette 500 µl GLUOX buffer into micro-centrifuge tube containing       pre-weighed glucose oxidase, vortex mix to dissolve,  add mixture remaining volume of buffer, stir, pour into staining container/coplin jar.   

                 B.  Use a 37°C water bath for even heating, NOT AN INCUBATOR with air currents causing uneven heating.          

Reference:    *Andrew SM, Jasani B.  An improved method for the inhibition of endogenous peroxidase non-deleterious to lymphocyte surface markers.  Application to immunoperoxidase studies on eosinophil-rich tissue preparations.  Histochem J 19:426-430, 1987

 

FYI:  Vector has this method in a free downloadable pdf brochure;  http://vectorlabs.com/brochures/   Multiple Antigen Labeling Guide p.20/Appendix 2/Method 3  

            I took the liberty of copying this from Vector who used mg terminology. 

            Method 3. 0.180 g b-D(+) glucose, 5 mg glucose oxidase, 6.5 mg sodium azide in 50 ml PBS.

            Incubate sections for 1 hour at 37 ºC. Rinse in PBS 3 x 5 minutes. This reaction slowly and steadily produces very low concentrations of H2O2 by enzymatic reaction. This method consistently and completely inhibits peroxidase activity.

            (Andrew S.M., Jasani, B.; Histochem J. 19, 426-430,1987.) 

 

Final comment.   If Patrick still has background staining after using Glucose oxidase method, then I suspect background is coming from another source which can be determined with an immunostaining reagent background test.   I will be happy to provide the simple background test  too. 

 

Take care

 

Gayle Callis

HTL/HT/MT(ASCP) 

 

 

Amos and Patrick Wrote: 

 

Hi,

     Peroxidase can really be a pain. If you look in the archives though

(or ask her really nice) Gayle Callis submitted a recipe for a glucose

oxidase for peroxidase quenching that does not include hydrogen peroxide.

If you aren't really a fan of making these things up I would bet dimes to

doughnuts that the recipe is *really* similar to the product from Biocare

Medical called PeroxAbolish.

Here's a link... http://biocare.net/product/peroxabolish/

I have used it and it did work, but I don't really use it regularly so

can't really compare it well. Incidentally if I am wrong in my assumptions

about the similarity of this to the glucose oxidase, I trust someone here

(even from the company itself) will gently correct me.

 

Cheers,

Amos

 

On Sat, Sep 12, 2015 at 1:00 PM, <histonet-request at lists.utsouthwestern.edu <http://lists.utsouthwestern.edu/mailman/listinfo/histonet> >

wrote:

 

> Message: 5

> Date: Fri, 11 Sep 2015 20:33:45 +0000

> From: "Lewis, Patrick" <patrick.lewis at seattlechildrens.org <http://lists.utsouthwestern.edu/mailman/listinfo/histonet> >

> 

> Hi Everyone

> 

> Thanks for your responses.

> 

> I am looking at cell surface markers,

> Sorry I should have said.

> 

> Based on what I found out below:

> 

> Methanol is out, even though I agree that Methanol does enhance the effect

> of H202 blocking.

> (I suppose I could try it to see how much/if any epitope loss there is in

> relation to H202 quenching,  At least It would help identifying false

> positives that are actually H202 artifacts.)

> 

> Also it looks like increasing the concentration of H202 is out.

> 

> As to when though,

> 

> It looks like with cell surface markers I should block after the primary,

> or even after the 2ndary?

> Can I do that successfully with a HRP labeled 2ndary?

> 

> thoughts?

> 

> Patrick.