Immunohistochemistry

** Paraffin or frozen sections for immunohistochemistry

Question.

Are paraffin or frozen (fixed) sections are better for IHC?

I’ve had great success in the past with frozen or vibrating

microtome sections, and have been trying paraffin lately,

but haven’t got any good results.

Answer 1.

Generally frozen sections are better for IHC because the

antigenic content is well preserved (provided the tissue is

snap frozen rapidly, preferably in isopentane, then stored

at -70C). A “good” frozen section cut at about 5 microns

should provide adequate morphology.

The advantages of paraffin tissue blocks is that larger

pieces of tissue can be used, and morphology is a degree

better, storage is easier, etc.

The disadvantage of paraffin blocks is the fact that the

processing of the tissue (especially when preserved in

common fixatives such as formalin or other formaldehyde-

based solutions) cross-links certain proteins in and on

the cells. Preatreatment to “unmask” cross-linked antigens

is often essential. Antigen retrieval techniques include

microwaving in citrate buffer and pressure cooker techniques.

However, some antigens are destroyed by paraffin processing,

so for these the manufacturer of the antibody should

recommend the use of frozen sections only.

Stephen Wayne

Cambridge Antibody Technology

The Science Park, Melbourn,

Royston, Cambridgeshire SG8 6JJ

England.

(stephen.wayne[AT]camb-antibody.co.uk)

Answer 2.

In general, immunoreactivity is often better in cryostat

sections than in wax sections, however tissue morphology is

usually not as clear. If you are getting satisfactory results

with cryostat sections, then I would probably recommend sticking

with that technique. However, if need to use wax sections for

whatever reason, there are several ways of tweaking the protocal

to try and improve the staining. Any good IHC text book will

outline most of these.

Off the top of my head, I would suggest playing around with the

fixation conditions or trying some form of antigen unmasking

step (particularly if you are currently seeing no specific

staining at all).

Ian Jones, PhD

School of Biological Sciences,

Queen Mary and Westfield College,

University of London, England.

(I.W.Jones[AT]qmw.ac.uk)

** Inhibiting endogenous peroxidase

Questions.

1. What is the best way to inhibit endogenous peroxidase

activity before doing an immunohistochemical method?

2. How long can methanol/H2O2 mixture (for quenching

endogenous peroxidases during IHC) be kept? or should

it be freshly made each time before use?

Different people favour different methods! Here are five

suggestions. All are claimed to work well, so probably

you should start with whatever you think is the easiest

and cheapest.

Answer 1.

We use a homemade version: PBS with 0.03% hydrogen peroxide,

and 0.1% sodium azide. Very gentle; doesn’t knock sections

off slides (frozens); can make up a one-week supply.

Use it once, then discard (we use dropper bottles).

Our PBS is at pH 7.4. We collect the leftover for chemical

disposal of sodium azide.

OR you can purchase DAKO peroxidase blocker with 0.03% H2O2

This block works best with our mouse antibodies as it does

not interfere with some of the IHC staining/per recommendation

of PharminGen. They use DAKO also, and if there are capillary

gaps involved, this does not produce the crummy bubbles that

drive one crazy.

Gayle Callis

(uvsgc[AT]msu.oscs.montana.edu)

Answer 2.

We prepare 600ml vats of methanol/H2O2 for use on a DRS601

and replace these weekly. It’s left on the machine for 5

working days then dumped. We’re handling about 150 ICC

slides/day.

Elwyn Rees

(100131.74[AT]compuserve.com)

Answer 3.

Just a personal note on the use of methanol in blocking

solutions; I have also found that methonal can be harmful

to some antigens, both hemopoetic and some infectious

disease antigens. We have found that performing our

endogenous peroxidase inactivation prior to any antigen

retreival step (either enzyme digestion or heat induced)

works best. For antigens sensitive to methanol and frozen

sections we use PBS containing 0.1% Na azide and 0.5% H2O2

with excellent results. Just be sure to wash the slides

well after this step because the Na azide is a potent

peroxidase inhibitor which will eliminate any specific

staining quite well. Using poly lysine coated slides will

generally keep frozen sections from lifting off.

Brian J. Chelack

(chelack[AT]admin3.usask.ca)

Answer 4.

Quenching with the glucose oxidase method works very

well, and is very gentle on sections, particularly frozen

sections. The only drawback is a bit more preparation of

solutions, but in the long run is a very COMPLETE quenching,

better than hydrogen peroxide, according the original

publication and method. I highly recommend it.

Gayle Callis

(uvsgc[AT]msu.oscs.montana.edu)

Answer 5.

Complete inhibition of endogenous peroxidase (including

activity in leukocytes and erythrocytes) can be achieved

by treating formaldehyde- or acetone- fixed smears or

sections with 0.024 M hydrochloric acid in ethanol for

10 minutes. To make this, add 0.02 ml of concentrated

(12 M) hydrochloric acid to 100 ml of ethyl alcohol.

Reference:

Weir EE + 4 others (1974) Destruction of endogenous peroxidase

activity in order to locate antigens by peroxidase-labeled

antibodies. J Histochem Cytochem 22:51-54.

This simple method doesn’t seem to be much used. I have tried

it, and Yes, it did work.

John Kiernan

(kiernan[AT]uwo.ca)

** Using mouse primary antibodies on mouse tissues

Question.

Using a mouse monoclonal on sections of mouse tissue often

makes a strong background staining because the secondary

antiserum binds to mouse immunoglobulin already present

in the tissue. Is there a way to get round this difficulty?

Answer(s) 1.

Two published methods seem quite good for this purpose.

They are very briefly summarized below. For practical

details consult the original papers:

Hierck,BP; Iperen,LV; Gittenberger-de Groot,AC; Poelmann,RE

(1994): Modified indirect immunodetection allows study of

murine tissue with mouse monoclonal antibodies.

J. Histochem. Cytochem. 42(11, Nov), 1499-1502.

Mouse monoclonal reacted with HRP-rabbit anti-(mouse serum);

then add excess normal mouse serum & incubate with tissue.

Lu,QL; Partridge,TA (1998): A new blocking method for

application of murine monoclonal antibody to mouse tissue

sections. J. Histochem. Cytochem. 46, 977-983.

Blocking with mixture of Fab and Fc fragments from

rabbit anti-mouse antibody. (Made by papain digestion,

then more Fc added). Stops background staining of

endogenous mouse IgG by the secondary antiserum.

Corazon D. Bucana, Ph.D.

Houston, Texas

(bucana[AT]audumla.mdacc.tmc.edu)

John A. Kiernan

London, Canada

(kiernan[AT]uwo.ca)

Answer 2.

[ This answer does not really explain what to do, but the

advertised product might interest users of mouse monoclonals.]

DAKO just released an immunostaining system for animal tissues.

In particular, it excels with mouse antibodies on mouse tissue.

We engage a novel technology to ensure clean background and high

specificity. Stoichiometric amounts of primary-antibody complex

are preformed before it is exposed to the tissue site. This

eliminates the unwanted reaction between secondary antibody and

mouse tissue.

Please visit the DAKO Corporation website (www.dakousa.com) to

request literature on the new DAKO ARK (Animal Research Kit). We

presented a poster at the IAP meeting in Boston and this

document is available by mail.

A few highlights: 1. One kit for all animal IHC testing utilizing

mouse monoclonal primary Abs. 2. Use on tissue from any animal

species. 3. Unique process eliminates background staining.

4. Staining results in 45 minutes. 5. Automatable

For more information please contact DAKO Technical Services at

techserv[AT]dakousa.com or call 800-424-0021.

Bret Cook

Product Specialist, DAKO Corporation

(general[AT]silcom.com)

** Antigen retrieval: A patented or copyright phrase?

Question:

I was talking to someone the other day concerning

immunoperoxidase staining and I mentioned the term “antigen

retrieval”. I was told that the term is patented and that it

was not legal to use the phrase. Has anyone else heard that

information. I do know that Biogenex makes and sells

“Antigen Retrieval Solution,” and we use it in our lab.

Is it really true that we cannot talk or write about antigen

retrieval in a general way without the risk of being sued for

some infringement of a copyright or a patent?

Answer.

This was the subject of some heated discussion in the

HistoNet listserver in 1998. The following remarks are

based on the contributions of people too numerous to

acknowledge individually, and are colored by my own

conclusions.

On the one hand there were the “common sense” viewpoints

making the case that

(a) A combination of two common words could not possibly

amount to an original literary composition (with

copyright assignable to an author or publisher), and

could never be construed as an invention. (A particular

solution could, of course, be invented for the purpose

of retrieving antigens, and patented.)

(b) Methods for enhancing the detection of antigens in

sections have been published in the scientific

literature for several years. All involve treatment

with water, which may be cold or hot, and most

techniques specify other substances to be dissolved in

the water. The solutes include detergents (to damage

cell membranes, helping large antibody molecules to

enter cytoplasm), urea (disturbs protein conformation

and may expose “buried” epitopes), a variety of metal

salts, notably zinc sulfate and lead thiocyanate

(probably work by changing the conformation of the

antigen), and all sorts of buffers, mostly pH 5-6 or pH

8-9. (This probably catalyzes hydrolysis of the

cross-links that formaldehyde makes between nearby

parts of protein molecules. The optimum pH varies with

different antigens. Heat accelerates the reaction, and

can be conveniently delivered in a microwave oven.)

On the other hand (Would it be the Left or the Right?)

were people using these methods daily, in routine

procedures, sometimes with a proprietary solution and

sometimes varying the technique to suit the antigen.

Feeling their freedom of expression (and perhaps also

their livelihoods) threatened, they suggested alternatives

to “antigen retrieval.”

The word “unmasking,” which has a long and honorable

history among histochemists, is a conspicuous improvement

on “retrieval” because it says what happens. The epitopes

of antigens were not retrieved (= brought back), because

they were already there. The hot water and other chemicals

made them accessible to the primary antibody by removing

physical and chemical barriers (“masks”) to the diffusion

of large molecules.

BUT people are human and by nature conservative (= change

can only make things worse), so it’s likely that

“retrieve” will win out over “unmask” despite any logical

arguments. The HistoNet discussions ended when Biogenex

said that the firm did not claim exclusive ownership of

the “antigen retrieval” word pair, and we could say or

write it without being sued.

John A. Kiernan, MB, ChB, PhD, DSc,

Department of Anatomy & Cell Biology,

The University of Western Ontario,

LONDON, Canada N6A 5C1

(kiernan[AT]uwo.ca)

** p53 protein

Question.

What is the significance of immunostaining with

antibody to p53?

Answer.

First of all, p53 is the antigen in the tissue, with which

the antibody combines (The p is for “protein”). p53 is also

sometimes referred to as a TSG – Tumour Suppressor Gene).

p53 was labelled “Molecule of the Year” by either Science or

Nature about three years ago.

The “wild” type p53 is the normal. It suppresses cell

transformation and/or mutations. It was traditionally

considered to have a very short life and was therefore never

present in concentrations large enough to demonstrate

immunocytochemically. “Mutant” type p53 has a longer “half-life”

and is therefore more easily demonstrated. It used to be that

mutant type p53 was the antigen of interest. Then of course,

things got more complicated.

There are, of course, antibodies to each type of p53 now.

One thing is for sure – p53 is of fundamental importance in

cell transformation. The biggest problem is that many consider

that the expression of p53 is quantitatively related to prognosis

and can therefore, be used to assess treatment outcomes. Whether

quantitation should be by percentage of positive (?tumour) cells

or by intensity of staining in the positive (?tumour) cells is

still open to debate. Whichever it is, it is obviously important

that your results of today can stand statistical comparison with

your results of yesterday or tomorrow. Even more importantly, can

they be used for comparisons with other labs? The patient may

move elswhere for treatment, for example.

One thing I know for certain: it is very easy to make virtually

all cells p53-positive – not just tumour cells – if you tweak your

immunocytochemical method and any heat induced antigen retrieval

you use. A real minefield!

Russ Allison, Wales

(Allison[AT]cardiff.ac.uk)

** Prevention of fluorescence fading

Question.

What is available in the way of chemical additives to aqueous

mounting media, commercial or homemade, to suppress fading of

immunofluorescence preparations?

Answer 1.

Jules Elias has a discussion about this in his book

“Immunohistopathology, A practical approach to diagnosis.” ASCP

Press, 1990. He says 1 percent p-phenylenediamine added to the

mounting medium retards fading.

Two references he gives:

Johnson, GD, et al, A Simple Method of Reducing the Fading of

Immunofluorescece During Microscopy. J Immunol Methods

43:349-380, 1981.

Huff, JC, et.al., Enhancement of Specific Immunofluorescent

Findings with use of para-phenylenediamine mounting buffer.

J Invest Dermatol 78:49, 1982.

Tim Morken

(timcdc[AT]hotmail.com)

Answer 2.

Look into Vectashield, it is supposed to a good mounting media

for immunofluorescence. You may not be able to prevent fading

entirely, because the exciting light can cause it. Storage of

the slides, after coverslipping, should be dark, sometimes in

cold, or even in a freezer.

Vectashield is from Vector and it is pricey: $40 for 10 ml.

Gayle Callis

(uvsgc[AT]msu.oscs.montana.edu)

Answer 3.

I think that the anti-fade agents that have already been

mentioned are all good, I must admit I have never used

Vectashield so will not comment on this. However, no mention

has been made of the possible variability in results with these

materials. Most of the anti-fade agents I have tried vary

considerably in their effectiveness. This appears to depend on

the specific antibody used, the fluorescent marker, the

fluorescence ratio of dye to marker molecule, whether the IHC

is direct or indirect and if you remembered to feed your cat

before going to work. As an example using lectin labelling of

cells with direct or indirect techniques, I found that the FITC

label was usually retained for UEA-1 but not for WGA. I would

therefore urge anyone who is going to use anti-fade agents to

try them first on some extra slides to test their

effectiveness.

Barry Rittman

(brittman[AT]mail.db.uth.tmc.edu)

** Background in immunostained cartilage

Question.

I have tried to immunostain sections of whole mouse embryos with

several primary antibodies to a nuclear epitope. I am getting

nonspecific antibody staining in cytoplasm and in the connective

tissue around the cartilage.

I have blocked with embryo powder, normal goat serum, normal

horse serum, beat blocking solution from Zymed, and Fab

fragments. What could be reacting with secondary alone?

Answer 1.

I do a lot of cartilage and bone IHC markers, mostly on rat, but

have done some mouse tissue. Is your primary made in a mouse?

Even with rat tissue, anti-mouse secondaries can combine

non-specifically with the rat tissue, I put rat serum in my

detection and it helps tremendously with the background.

Patsy Ruegg

(rueggp[AT]earthlink.net)

Answer 2.

The different blocking steps you have tested all block

hydrophobic areas (“sticky sites”) in your specimen. Hydrophobic

areas are blocked before the immunoincubation with e.g. normal

serum or BSA. Once blocked these sites generally will not give

rise to background anymore.

Cartilage and perichondrium are composed of collagen fibers with

a positive charge (still present after aldehyde fixation)

embedded in proteoglycans which have a negative charge. Most

antibodies (primaries and secondaries) are negatively charged at

pH 7-8.2. I therfore think that the collagen fibers present in

the cartilage tissue are causing your background problem. This

charge-determined background can be circumvented by

adding negatively charged molecules (e.g. aurion BSA-c) to the wash

and incubation buffers. Another possible cause for background

(a specific binding to proteoglycans) can be prevented by adding

gelatin to your buffers. Do not put both BSA-c and gelatin in

the same buffer, because they have charge-determined affinity

for each other as well.

I invite you to visit our web-site for detailed info on the

topic above. http://www.aurion.nl

Peter van de Plas

AURION,

Wageningen, Netherlands

(vandeplas[AT]aurion.nl)

** Endogenous biotin in mast cells?

Question.

Do mast cells contain any endogenous biotin? They are often

falsely positive in immunostaining methods that use avidin.

Answer 1.

Mast cells bind avidin nonspecifically because of ionic attraction

between avidin (a basic protein) and heparin (acid polysaccharide

in MC granules). This results in false positive staining by ABC.

The cure is to use the ABC reagent at pH 9.4. For more information

see Bussolati, G & Gugliotta, P 1983. Nonspecific staining of mast

cells by avidin-biotin-peroxidase complexes (ABC). J. Histochem.

Cytochem. 31: 1419-1421.

John A. Kiernan, Department of Anatomy & Cell Biology,

The University of Western Ontario,

LONDON, Canada N6A 5C1

(kiernan[AT]uwo.ca)

Answer 2.

Bussolati and Gugliotta (J. Histochem. Cytochem., 31(12):

1419-1421, 1983) described binding of ABC to mast cells.

They believed this to be due to both the binding of

avidin basic residues as well as peroxidase to the sulphate

groups of heparin. They showed that binding could be prevented

by using the ABC solution at a pH of 9.4. This high pH does

not affect either previous binding or localisation of antibody

or the affinity of biotin for avidin.

They also showed that the nonspecific binding of avidin

could be blocked by a 30 minute pretreatment of sections with

a synthetic basic polypeptide such as poly-L-lysine (0.01%

in PBS, pH 7.6).

Tony Henwood, Senior Scientist

Anatomical Pathology

Royal Prince Alfred Hospital

Sydney, AUSTRALIA

http://www2.one.net.au/~henwood

http://www.pathsearch.com/homepages/TonyHenwood/default.html

(henwood[AT]mail.one.net.au)

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