Keele, JA 1

Immunohistochemistry in the Heart: A Protocol Manual

By: Jacque A. Keele and Mark M. Stayton Department of Molecular Biology, University of Wyoming,

Laramie, WY, 82071, USA July 2008

IMMUNOHISTOCHEMISTRY IN THE HEART: A PROTOCOL MANUAL....... 1

PART I: INTRODUCTION AND BACKGROUND .................................................................... 3

PART II: MATERIALS AND METHODS ............................................................................. 19

PART III: DEFINITIONS- FROM THE HANDBOOK IMMUNOCHEMICAL STAINING METHODS

3RD EDITION, DAKO CORPORATION (1)......................................................................... 23

REFERENCES .................................................................................................................. 26

Figure and Table List Figure 1: Diagrams of the binding of (A) polyclonal and (B) monoclonal antibodies to

epitopes of an antigen. Note that polyclonal antibodies bind to many different

epitopes while monoclonal antibodies bind to a single specific epitope. (C) The

direct method where the primary antibody (green) is directly labeled with a tag (red

dot) to the protein of interest (blue). (D) The two-step indirect method of detection.

The protein of interest (blue) is first labeled with the primary antibody (green), the

secondary antibody (yellow) binds to the primary antibody, and a fluorescent tag

(red) is used for detection (1)...................................................................................... 9

Figure 2: Autofluorescence in a chemically fixed heart section. A) TRITC filter, B) FITC

filter, C) merged image, 20x magnification.............................................................. 17

Figure 3: Autofluorescence of naïve heart tissue. (A) TRITC filter, (B) FITC filter, (C)

Merged, (D) Dapi...................................................................................................... 17

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Figure 4: 40x Olympus microscope images of ARG2, two different slides. (A) ARG2

with stats, (B) Dapi, (C) merged, (D) ARG2 without stars, (E) Dapi, (F) merged... 18

Figure 5: Naïve expression of N-cadherin (1:2 dilution) of mouse heart using the

Olympus microscope 40X magnification. Changing the exposure time of the digital

camera can result in under or over exposure of the image. (A) Under exposed, (B)

Middle exposure time, (C) Overexposed .................................................................. 18

Figure 6: Selecting an image for publication. Double labeling of ARG1 (red) and SPSY

(green) in naïve mouse heart at 40X magnification.................................................. 18

Figure 7: A) Humidity control box with wet paper towel. B) Microscope slide with the

microscope slide holder in it. Note that one of the microscope slides has PBS on it

(volume approximately 150 µL). C) Washing the slides with PBS. D) Microscope

slide with the cover slide sealed on by nail polish.................................................... 22

Table 1: List of useful websites and suppliers for heart immunohistochemistry ............... 6

Table 2: Comparison of cryopreservation and chemical fixation processes for the

preparation of samples for immunohistochemistry..................................................... 8

Table 3: Phosphate Buffered Saline (PBS)....................................................................... 19

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Part I: Introduction and Background

Scientists have applied immunohistochemistry (IHC) to a wide variety of

organisms, tissues, and cells. The focus of this manual will be on mouse heart because it

is the tissue most studied in the Stayton Lab. The goal of the experiments performed in

the Stayton Lab was to determine the naïve expression pattern and subcellular location of

specific proteins in the heart. In addition, once the naïve expression pattern of the

proteins of interest were determined it was possible to observe changes in expression

patterns following the application of surgical acute myocardial infarction on the heart.

There are many different approaches to performing IHC; therefore, this manuals focus

will be on the general background information, the protocols, and troubleshooting

problems seen when working on mouse hearts. While the focus of this manual is on

mouse heart tissue, the background information in it can be applicable to other tissues

with modifications. The ultimate goal for anyone performing IHC should be

reproducible, reliable results (the three R’s). Obtaining this goal can be a challenge, but

it is possible to do when there is a basic understanding of the IHC process. Often

obtaining this goal can require weeks or sometimes months of trial and error to determine

the optimal conditions for tissue processing, staining, and microscope observations.

Immunohistochemistry (IHC) involves the study of cells and tissue.

Immunocytochemistry applies to the study of the intracellular activities of proteins.

These two words are now being used interchangeably in scientific publications and

presentations. Both of these techniques use antibodies to detect specific antigens in a

sample has been placed on a microscope slide.

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IHC is very similar to the method used for western blotting; both of these

methods involve blocking, primary and secondary antibody incubations, and finally

detection as key steps. Another technique that is gaining popularity is the use of green

fluorescent protein (GFP) labeled proteins. For this method, a transgenic animal is

produced that expresses GFP-fusion protein in the animal. The advantage of this method

is that tissue can be harvested from these animals, sectioned, and observed on the

microscope without IHC staining. The GFP will show you where your protein of interest

is located assuming that GFP does not alter the protein localization. While technique is

commonly used in yeast and C. elegans, using it in mice would require the production of

a transgenic animal. The cost of producing a GFP-labeled protein in a mouse when

compared to immunohistochemistry staining is high.

Using the Internet to Find Protocols

The internet is an amazing starting place for finding protocols, antibodies, and

supplies needed to perform IHC (Table 1). It is important to the researcher to find out as

much background information as possible on the antibodies and protocols they will be

using. For starters, you should find out if others have used the antibody for IHC. If they

have, then did they work on the same tissue or organism that you are interested in

studying? If they did, then using the protocol that they developed for the antibody is a

viable starting place for your own experiments. If there are not published data on your

antibody and organism the next step is to determine how others have prepared the sample

you are interested. By looking at how others have approached IHC with your sample,

you can avoid some of the problems and time involved in developing your own protocol.

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Protocols that are more detailed than those published in journals are available from the

antibody suppliers and from the website www.IHCworld.com.

Second, realize that getting a published report on both your antibody and sample

of interest can be a challenge. The majority of antibodies used in the Stayton Lab to label

proteins in the heart had very little data available as a starting place. In that case, we had

to develop our own protocols based on the IHC that others had done in the heart and

work our own conditions for staining for each of the antibodies.

Finally, if you purchase commercial antibodies they should come with a data

sheet that will show the methods the antibody can be used for, dilution factors, storage

conditions, and any publications in which the antibody was used. Before purchasing an

antibody, it is important to look at these data sheets and determine if the antibody that

you are thinking of purchasing is usable for IHC and compatible with the sample you are

interested studying. In addition, the data sheet can tell you if there are any special

considerations, you need to make when using the antibody (i.e. buffers, incubation times,

fixed or unfixed tissue).

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Table 1: List of useful websites and suppliers for heart immunohistochemistry

Key Steps in IHC Tissue Fixation There are two ways to prepare tissue for IHC- chemical fixation or cryofixation

(Table 2). Chemical fixation uses cross-linking chemicals such as para-formaldehyde

and gluataldehyde to preserve cellular structure. Once the tissue is harvested, the fixation

begins. This process can be done by hand or by specialized instruments. Following the

fixation, a tissue block is made by placing the sample in hot parafilm, placed into a mold,

allowed to cool and harden, and thin tissue sections can then be made. These slides can

be stored at room temperature.

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The thin sections on the slides need to be “de-cloaked’, so that antigens are

exposed. De-cloaking methods include heat and pressure treatment, enzyme digest, and

microwaving. Following the de-cloaking, the parafilm on the slides is removed by

baking, and the IHC staining process can begin. With these slides, autofluorescence is a

major concern and de-cloaking the epitopes can be difficult (Fig 2).

Cryofixation does not preserve the cellular structure quite as well as chemical

fixation, but the amount of autofluorescence seen with cryo-fixed tissue is substantially

less than the chemically fixed tissue. Cryofixation for heart muscle (or for any other type

of muscle) is done by using iso-butane cooled by liquid nitrogen. Hearts are placed in a

small mold or boat, and O.C.T. (Optical Cutting Temperature) is pored over the heart.

The boat is then dipped into the cold iso-butane, and the sample is cooled almost

immediately. Once the samples are in this block, they must be stored at -80oC until they

are sectioned. The O.C.T. helps to protect the tissue during the freezing process. Using

this method ensures that the sample is frozen as quickly and thus decreases the likelihood

of ice crystals forming that can damage the tissue.

Tissue sectioning of the heart is performed on a cryostat set at -18oC to -20oC.

Keeping the cryostat as cold as possible is key to obtaining good sections. When the

cryostat gets to warm, the O.C.T. will melt becoming gummy, and it will become

impossible to get clean sections. After the slides are made, they should be set out to air

dry for at least 10 minutes, and then stacked, wrapped in tinfoil, and stored at -80oC until

needed. Sections can be stored for long periods at -80oC if they properly protected.

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Table 2: Comparison of cryopreservation and chemical fixation processes for the preparation of samples for immunohistochemistry.

Antibodies

Antibodies can be used in a multitude of different experiments. For example,

antibodies are used in: immunohistochemistry, western blotting, radioimmunoassay or

and ELISA, protein purification, gel shift experiments, and flow cytometry. Before

beginning any experiment, it is important to know if the antibody being used will work

for the experiment you are proposing to perform. Some antibodies will work for

immunohistochemistry and not so well for western blotting, and visa versa. Therefore, it

is important to know the characteristics of the antibody. In addition, it is important to

determine if the antibody will work with the sample that you wish to study.

Commercially available antibodies will have a data sheet that will list the recommended

applications and samples that the antibody has been tested for.

There are two different types of primary antibodies used in

immunohistochemistry: monoclonal and polyclonal. Monoclonal antibodies are made

using cell lines (usually mouse) and recognize one epitope on the antigen (Fig 1).

Polyclonal antibodies are made by injecting a protein of interest into an animal (rabbit,

goat, ect), the animal has an immune response to the protein and produces antibodies

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against the protein. The serum is harvested and the antibodies are purified. For both

monoclonal and polyclonal antibodies, it is important to determine the optimal working

dilutions before starting to collect real data.

Detection of the protein is done directly with the primary antibody or indirectly

with a primary antibody that binds to a secondary antibody (Fig 1 c and d). The primary

antibody can be conjugated to a fluorescent tag. There are kits are available for

conjugating primary antibodies to tags. The more common way of detecting epitopes is

to use a labeled secondary antibody that will bind to the protein of interest.

Figure 1: Diagrams of the binding of (A) polyclonal and (B) monoclonal antibodies to epitopes of an antigen. Note that polyclonal antibodies bind to many different epitopes while monoclonal antibodies bind to a single specific epitope. (C) The direct method where the primary antibody (green) is directly labeled with a tag (red dot) to the protein of interest (blue). (D) The two-step indirect method of detection. The protein of interest (blue) is first labeled with the primary antibody (green), the secondary antibody (yellow) binds to the primary antibody, and a fluorescent tag (red) is used for detection (1). Secondary antibodies can be conjugated to an enzyme, biotin, or fluorescent tag.

This manual will focus on fluorescent labeling. When enzymes labeling is done, the

secondary antibody has to undergo a chemical reaction to produce a color product.

Secondary antibodies are usually absorbed against control animal serum to reduce

background staining. Choosing the correct secondary antibody is key to having the

experiment work out well. First, decide how the antibody will be detected, will it be with

fluorescence or the light microscope. Then pick the secondary antibody. There are

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companies that specialize in secondary antibodies (Jackson Immuno Research for

example) and have their catalogs posted on line. The variety of different secondary

antibodies can be overwhelming. Companies selling the primary antibodies will usually

have a data sheet where they tell you the recommended secondary antibody to use. In

addition, it is also advisable to call the company’s technical support and get an experts

opinion about the secondary antibody before making the purchase.

It is important plan out IHC experiments prior to purchasing antibodies for several

reasons. First, antibodies are expensive (primary antibodies start at $200 and go up) and

you do not want to buy antibodies that are not compatible with your samples. Second, if

multiple antigen labeling is going to be done it is important to know if the antibodies are

compatible with each other. Third, it is possible to shop around for your antibodies to get

a better price because there are many companies selling primary antibodies. Finally, take

the time to read up on the antibodies that you will buy. Some antibodies work only under

certain conditions (blocking buffer, antibody dilution, and ect.) and it is important to

know these details prior to using the antibody. Purchasing antibodies is an investment

and like all good investments, it is very important to do background research.

Antibodies should be stored according to the manufacturer’s recommendations.

Usually it is advised that the antibodies should be stored at 4oC to avoid freezing and

thawing cycles. It is also possible to aliquot the antibody for long-term storage at -80oC

using instructions provided by the manufacturer.

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Immunohistochemistry Staining In Part II, of this appendix, the protocols used for double labeling are laid out in

detail. However, in every protocol, minor details or lessons learned sometimes are left

out that can help to make IHC process work more efficiently. This section will discuss

some of the do’s, don’ts, and why’s of IHC that might seem like common sense, but that

can be forgotten in the rush to get results.

First, it is always important to work with fresh buffers and solutions. The

antibody and blocking buffers should be made fresh every few days to ensure that the

components in these buffers do not degrade. This can lead to a decrease in the quality of

the IHC staining. Second, it is important to determine the optimal antibody dilution for

the experiments. After looking at several different dilutions, you determine which of the

dilutions gives the “best” staining. Finally, the washes done with PBS after the primary

and secondary antibody incubations are very important. If they are not sufficiently

stringent, there can be over-staining, artifacts, and stars (see troubleshooting images).

Quality control is also an important factor to consider when setting up IHC

experiments. If possible, it is important to have a positive tissue control that will show if

the antibody is working correctly. Negative controls, where the primary antibody is

absent, are used to assess if the secondary antibody is binding non-specifically to the

tissue. If non-specific secondary binding is seen then changes in the blocking buffer

must be made. In addition, you want to have consistent results over time. If the

antibody-staining pattern changes every time you do an experiment, then something is

wrong with your experimental set up. The goal for anyone performing these types of

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experiments has to be reproducible reliable results (the three R’s) that anyone in the lab

can obtain.

Blocking is a vital step for IHC. Blocking buffer should contain serum from the

animal from which the secondary antibody was raised. This is done to avoid having the

secondary antibody bind non-specifically to the tissue. For example, if the primary

antibody was raised in a rabbit, then your secondary antibody will have to be anti-rabbit.

If the secondary is goat anti-rabbit, then you will need goat serum in the blocking buffer.

The composition of the blocking buffer can change depending on the antibodies that are

being studied. BSA can be in the blocking buffer; however, some secondary antibodies

will bind non-specifically to BSA creating a false-positive result. So with these

antibodies, BSA is omitted from the blocking buffer. Optimizing the blocking buffer can

help you avoid background staining and false-positive results. Knowing about these

simple yet very important details can help to ensure clean reliable reproducible IHC

results.

Microscope Observations, Photography, and Data Analysis

Books have been written about the ways to make microscope observations,

photography and data analysis. This section will briefly discuss several of the common

issues that were encountered and overcome when studying the mouse heart with IHC.

First, all tissue has some natural fluorescence associated with it (Fig 3). Therefore, when

working with the fluorescent microscope it is important to keep in mind that the tissue

will autofluorescence and might give misleading results. To control for this, a slide

without any antibodies on it should be observed to assess the natural fluorescence of the

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tissue. In particular, it is important to determine if the autofluorescence is located in a

specific place or is through out the tissue sample.

When making microscope observations it is important to understand what the

unstained sample should look like. Knowing this will help you determine if your sample

looks normal or abnormal when compared to the published images. The process of

cryofixation and then sectioning can damage the tissue. Sectioning can tear the tissue,

the knife can cause ridges and nicks in the sample, and tissue can become folded.

Additionally, there can be trash (hairs, dust, and ect.) on the slide that can cause

interference with the analysis. Sometimes the trash will be fluorescent and give false

results. These are all issues that can be hard to prevent, so it is important to be aware of

them.

There are some key controls that should be done to determine the background

fluorescence on the slides that are being stained. First, a slide without any antibodies

should be done to show the natural fluorescence associated with the tissue of interest.

Second, it is important to determine if the secondary antibody is binding to the tissue. If

this happens adjustments in the blocking buffer must be made to ensure that the results

are true and not due to non-specific binding of the secondary antibody.

It is important to know and understand the sample under observation so

judgments can be made on whether the cryo-fixation or sectioning damaged the tissue.

So finding published images of the sample you are looking at can help you judge if the

sample is undamaged by the steps that went into making the section. In addition, it is

important to use control antibody markers to identify the cell type the protein of interest

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co-localizes to. DAPI is also a valuable tool in determining where DNA in the cells is

located.

Once an image is taken, it should be saved with as much identifying information

as possible. For instance, the date, tissue type, antibody dilution, tissue treatment, and

magnification of the microscope are all important information to know that will make

analyzing the images easier. Never make changes to the original image file. When you

begin to analyze the images, always open the image and then do a save as. IHC images

can be analyzed using the free software from the National Institutes of Health, ImageJ

(http://rsb.info.nih.gov/ij/). The website that provides this free software has tutorials on

how to utilize ImageJ. This program will allow you to merge images for double labeling,

and to make adjustment with the brightness and contrast of the images. It is a valuable

tool for analyzing and making the images ready for publication. Quantification of the

signal in IHC images is a recent development, and should be approached with great care

and thought. The software and algorithms needed to perform quantitative

immunohistochemistry have yet to be fully developed (2).

Troubleshooting- Examples of When Things Go Wrong

The following figures illustrate some of the common things that can go wrong with

IHC. Some of these things cannot be avoided (autofluorescence, artifacts, folded tissue)

while others can be controlled (stars, over and under staining, camera exposure). One

simple way to avoid making a time consuming mistake is to start out by doing single

labeling of the antibodies under study. Once the conditions staining have been

determined then it is possible to move into double labeling. If complex multi-labeling

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experiments are attempted from the start, mistakes will be made and it will be hard to

diagnose where the missteps are in the experimental design.

The goal should always be clean reproducible reliable results (the three R’s). Taking

clear and complete notes on everything that is done to the sections is important, because

with IHC the little things really do matter. Slight changes in incubation times, buffers,

and antibodies can result in significant changes in the IHC results. Most antibody

companies have tables available for troubleshooting IHC on the internet. In addition, it is

possible to call and discuss troubleshooting issues with the company that you bought the

antibodies from.

Autofluorescence is always a concern. It is important to test the sample to

determine how much the sample fluoresces without any antibodies present. In Fig 3, the

autofluorescence for naïve mouse heart cryo-sectioned sample is shown. When these

images were taken the exposure time on the camera was set at maximum. The images

were processed using ImageJ software. In Fig 3-A and B, it is possible to see that the red

and green filters of the microscope show autofluorescence. The green channel is stronger

than the red channel as shown when the images are merged. When these images were

processed, the brightness and contrast of the picture had to be increased using ImageJ.

Unambiguous staining is shown in Fig 3-D with Dapi labeling. In this image, it is

possible to see what specific staining looks like when compared to autofluorescence.

Another issue that comes up with IHC is artifacts or damage to the tissue due to

sectioning. Artifacts can be very fluorescent and sometimes give false results. They can

also disrupt the analysis of the slide if they are present in a specific region of tissue you

are looking at. Damage to the sample by the sectioning process can result in folded

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tissue, rips or tears in the tissue, and knife marks. Artifacts are usually easy to see and

diagnosis.

One artifact that is harder to diagnosis is when there are small fluorescent dots on

the slide that are known as stars. These stars are the result of inadequate washes before

mounting of the sample. They are caused by unbound secondary antibody left on the

slide. To recognize stars, you look at an area on the slide where there is know tissue

section. If there are stars in that area, then you know that the pattern of staining on the

section is false and is not specific binding of your antibody. In Fig 4-A, shows a star

pattern with ARG2 staining. Fig 4-B shows what happened when the washes were made

more stringent.

Changing the exposure time of the camera can have drastic effects on the image

quality (Figure 5). Overexposing or underexposing the section can result in a loss of

information. Finding the right exposure time is vital for getting publication quality

images.

One of the tricky aspects of IHC is selecting an image for publication. Figure 6

shows naïve mouse heart that has been stained with ARG1 (red) and SPSY (green).

These images were analyzed using ImageJ. Looking at this selection of images it is hard

to pick an image that would stand up to the rigors of peer review. The majority of the

images have an inbalance in their color. In addition, there is some starring in figure 6-A

and 6-H. The only one that might be publishable is figure 6-D. When selecting an image

it is important to consider a few things. First, did the IHC staining process work

correctly? Can the results be reproduced on multiple occasions? The staining showed in

Fig 6 was the first attempt at double labeling these two antibodies, therefore the results

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are very preliminary and would not be published. Second, which image best represents

the data? How many images are should be taken to ensure that the staining pattern is

consistent over days and different sections of tissue? These questions should be

discussed within the lab and decisions should be made on how to address these issues.

Figure 2: Autofluorescence in a chemically fixed heart section. A) TRITC filter, B) FITC filter, C) merged image, 20x magnification

Figure 3: Autofluorescence of naïve heart tissue. (A) TRITC filter, (B) FITC filter, (C) merged image, (D) DAPI.

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Figure 4: 40x Olympus microscope images of ARG2, two different slides. (A) ARG2 with stars, (B) DAPI, (C) Merged, (D) ARG2 without stars, (E) DAPI, (F) Merged

Figure 5: Naïve expression of N-cadherin (1:2 dilution) of mouse heart using the Olympus microscope 40X magnification. Changing the exposure time of the digital camera can result in under or over exposure of the image. (A) Under exposed, (B) Middle exposure time, (C) Overexposed

Figure 6: Selecting an image for publication. Double labeling of ARG1 (red) and SPSY (green) in naïve mouse heart at 40X magnification.

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Part II: Materials and Methods Buffers and Chemicals for Immunohistochemistry (All of these solutions are adapted from http://www.IHC.com protocols) Table 3: Main Buffer Used in IHC- Phosphate Buffered Saline (PBS) PBS pH 7.2 10X- 0.1M 20X- 0.2 M Na2HPO4 (anhydrous) 10.9 g 21.8 g NaH2PO4 (anhydrous) 3.2 g 6.4 g NaCl 90 g 180 g Distilled water 1000 mL 1000 mL Store this solution at room temperature. Dilute with distilled water before use, adjust pH to 7.2, and autoclave the solution. Blocking Buffer Normal Serum Blocking Solution: 2% serum (blocking) 1

1% BSA (stabilizer) 2

0.1% cold fish skin gelatin (blocking) 3

0.1% Triton X-100 (penetration enhancer) 0.05% Tween 20 (detergent and surface tension reducer) 0.05% sodium azide (preservative) 4 0.01M PBS, pH 7.2 Mix well and store at 4 ºC. Be aware that every antibody is different and adjustments will most likely be needed for the blocking buffer. Antibody Buffer Primary Antibody Dilution Buffer: 1% BSA (stabilizer and blocking) 0.1% cold fish skin gelatin (blocking)5 0.05% sodium azide (preservative) 6

1 Use serum from the animal that your secondary antibody was raised in. For example, if you are using a goat anti-rabbit secondary antibody then you will block with goat serum. In addition, if you are double labeling, you will block with two different serums. 2 BSA can cause background when used with certain secondary antibodies, so be aware of your secondary antibodies instructions. 3 Knox gelatin can be used in place of fish skin gelatin. 4 By making a small amount of blocking buffer and using it within a week, you can avoid the use of sodium azide. 5 Using Knox gelatin instead of the cold fish skin gelatin does not seem to affect the results obtained. 6 By making fresh dilutions of the primary antibodies right before they are used the addition of sodium azide can be avoided.

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0.01M PBS pH 7.2 (TBS pH 7.6 used in primary antibody dilution buffer produces weaker staining) Antibodies diluted using this buffer can be stored at 4 ºC for 6 months without reducing binding activity. This buffer cannot be used for diluting HRP conjugated antibodies since sodium azide is an inhibitor of HRP. Secondary Antibody Dilution Buffer: 0.01M PBS, pH 7.2 0.05% sodium azide (preservative) 7

Antibodies diluted using this buffer can be stored at 4 ºC for 6 months without reducing binding activity. Do not use BSA or other serum containing reagents to dilute secondary antibodies since they may bind to BSA or serum therefore reducing antibody affinity. Using TBS to dilute secondary antibodies often produces weaker staining. So use TBS only for the antibodies with high background staining or for alkaline phosphatase conjugated antibodies since phosphate is an inhibitor of alkaline phosphatase.

Anti-fade Mounting Medium8 2% n-propyl gallate 9 49% PBS pH 7.4 49% glycerol 0.5-1.0 ug/mL Dapi (final concentration)10

7 By making fresh dilutions of the secondary antibodies right before they are used the addition of sodium azide can be avoided. 8 Store the mounting medium at -20oC, wrapped in tin foil. It is light sensitive and will last longer if properly. From Dr. Z.J. Zhang, University of Wyoming 9 To prepare a stock solution of n-propyl gallate: 0.1M solution in 9:1 glycerol: PBS then place 0.1 g n-propyl gallate into 5 ml of glycerol: PBS. 10 Dapi can be left out of the anti-fade medium.

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Protocol for Double Labeling with Immunohistochemistry Parallel Approach (Adapted from http://www.IHC background/Immunofluorescence Double Staining Protocol.htm) Prior to perform double labeling, it is important to test each primary antibody individually and select the best pretreatment for each antibody (3). It will be ideal if the two primary antibodies require same pretreatment. Otherwise, one should do a further test by treating sections with both pretreatments and then immunostain for each antibody individually. If both antibodies survive the “double pretreatments”, you are ready for immunohistochemistry double staining. Overall IHC Method11 -Frozen Sections

a. Snap frozen fresh tissues in isopentane pre-cooled in liquid nitrogen, embedded in OCT compound in cryomolds. Store frozen blocks at -80 ºC.

b. Cut 4-8 µm-thick cryostat sections and mount on superfrost plus slides, gelatin-coated slides, or 1% silane coated slides. Store slides at - 80 ºC until needed.

c. Before staining, warm slides at room temperature for 30 minutes and fix in ice-cold acetone for 5 minutes. Air-dry for 30 minutes.

d. Wash in PBS for 2 x 2 minutes -Blocking e. Incubate sections in normal serum blocking solution – species same as secondary

antibody for 20 minutes (for example: primary antibodies are mouse and rabbit, and secondary antibodies are horse anti-mouse, and goat anti-rabbit, so horse and goat serum block should be used).

a. Made sure that the blocking buffer covers the sections, it should take less than 100 µL of liquid.

b. Do not let the slides dry out. Place the slides in a closed container with a damp towel to maintain the humidly. (Figure 7a)

c. Place the slides on the metal rack (Figure 7b) -Primary Antibodies

f. Incubate sections in the mixture of two primary antibodies at appropriate dilution in primary antibody dilution buffer for 1 hour at room temperature or at 4oC overnight.

a. Make sure the antibody mixture covers the sample. It should take less than 100 µL of liquid to cover the sample.

g. Rinse in PBS for 3x2 minutes (raise the metal rack to allow the liquid to drain off Figure 7c)

-Secondary Antibodies h. Incubate sections in a mixture of the two fluorescent conjugated secondary

antibodies for 30 minutes at room temperature. Make sure to do this in the dark. i. Rinse in PBS for 3x2 minutes

11 Sources for the supplies and equipment are listed in Table 5; in addition, materials can be obtained from chemical and equipment supply companies (such as Vector, Sigma-Aldrich, or the on campus Chemical Stock Room).

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-Mounting j. Place anti-fade mounting media on the sample- about 10 µL will cover the

sample. k. Place a glass cover slide over the sample l. Use nail polish to seal the cover slide to the microscope slide by painting on at

least two of cover slides sides (figure 7d) m. Store slide in the dark at 4oC

Figure 7: A) Humidity control box with wet paper towel. B) Microscope slide with the microscope slide holder in it. Note that one of the microscope slides has PBS on it (volume approximately 150 µL). C) Washing the slides with PBS. D) Microscope slide with the cover slide sealed on by nail polish.

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Part III: Definitions- From the Handbook Immunochemical Staining Methods 3rd Edition, DAKO Corporation (1). Antigen- a molecule that is capable of binding to an antibody Antigen retrieval (AR) - also known by the terms “epitope retrieval” or “target retrieval”, pertains to the restoration of antigenicity (immunoreactivity) to an immungen Antiserum- a serum that contains antibodies Ascites or ascetic fluid- an accumulation of fluid in the abdominal cavity Background- unless defined otherwise, background staining includes all nonspecific staining because of procedural artifacts. Occasionally, it may also include “undesirable” staining, e.g. due to diffused antigen Chromogen- one of a group of chemical species that can form a particular colored material or can be identified by such a reaction with an appropriate reagent Counterstain- a second stain that provides a contrasting effect to another stain Cross-reactivity- the ability of an antibody to react with antigens other that the immunogen. The term should not be used when referring to reactions occurring between an antibody and different cell or tissue components. Epitope- the structural part of an antigen that reacts with an antibody. These are groupings of amino acids in globular proteins and sugar side-chains in polysaccharides. The most critical part is called the immunodominant point. Immunochemistry- the branch of immunology concerned with the chemical substances and reactions of the immuno system, the specific study of antigens and antibodies and their interactions with one another. Immunocytochemistry- immunochemistry applied to the study of intracellular activities. (Now frequently used interchangeably with immunohistochemistry.) Immunogen- any substance capable of generating an immune reaction, in contrast to any substance that binds to an antibody (i.e., an antigen) Immunogencity- the ability of an immunogen to elicit an immune response. Immunogencity depends upon foreignness to the host, the size of the immunogen, the complexity of its molecular structure, the length of time it remains in the host and its ability to reach certain immuno-competent cells in order to generate immunity.

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Immunohistochemistry- immunochemistry applies to the study of cells and tissues. (Now frequently used interchangeably with Immunocytochemistry.) In Situ Hybridization- an assay for nucleic acids “on site” in fixed tissue sections by the use of heat to first denature and then to re-anneal with specific DNA, RNA or PNA probes Internal tissue control-a specimen from the patient donor, which contains the target marker, not only in the tumor to be identified, but also in adjacent normal tissue. Thus, no separate positive control sections are needed Ligand-a molecule, ion or atom that is bound to the central atom (usually a metal atom) of a coordination compound or chelate. Monoclonal Antibodies –immunochemically identical antibodies produced by one clone of plasma cells that react with a specific epitope on a given antigen. Produced commercially using hybridomas Monospecific-having an effect only on a particular kind of cell or tissue, or reacting with a single antigen, as a monospecific antiserum Negative tissue control- a tissue specimen from the same organ lacking the target antigen and processed by use of the primary antibody. Nonimmune serum- serum obtained from animals that have not been immunized. Polyclonal antibodies- immunochemically dissimilar antibodies produced by different cells and reacting with various epitopes on a given antigen. Positive tissue control- a specimen previously shown to stain specifically for the target antigen after exposure to primary antibody. Nonspecific background staining should be at a minimum. Note that, for some target antigens (e.g., prostate specific antigen), the staining intensity ideally should be less than maximal to allow monitoring not only for positivity, but also for variation in intensity. Primary antibody- the first antibody used in a staining procedure. Quenching- refers to the inactivation of a chemical activity by an excess of reactants or products. In enzymology, excess substrate or product may inhibit the enzymatic activity. Secondary antibody- the second antibody used in a staining procedure; it reacts with the primary antibody, now the antigen, and forms a bridge between the primary antibody and a subsequent reagent, if any. Also known as “link” antibody. Specific staining- positive staining of tissue or cells by use of primary antiserum. Occasionally this includes diffused, absorbed or phagocytosed antigen, giving rise

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to “undesirable” staining. The staining seen due to contaminating antibodies in the primary antiserum should be considered as nonspecific. Standardization- classically, to standardize means to compare with or conform an assay of unknowns to established standards. In quantitative analytical work numbers readily allow for conforming to such standards. In semi-quantitative or qualitative assays such as immunocyto- or immunohistochemistry, which frequently conclude with an opinion, only subjective comparisons to carefully selected tissue and reagent controls can be used to monitor and maintain excellence Titter-in immunohistochemistry, the highest dilution of an antiserum which results in optimal specific staining with the least amount of background.

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References (1) Boenisch, T. (2001) Immunochemical staining methods, 3 ed., DAKO

Corporation, Carpinteria, CA.

(2) Matkowskyj, K. S., D, and Benya, R. (2000) Quantitative immunohistochemistry

by measuring cumulative signal strenght using commercially available software

photoshop and Matlab. The Journal of Histochemistry and Cytochemistry 48, 303-

311.

(3) Javois, L. Immunocytochemical methods and protocols, Vol. 115.