Modern Immunofluorescence Techniques

Lecture 7

Practical selection of primary and secondary antibodies

Single-, double- and triple immunolabeling experiments

Issues of antibody cross-reactivity and host specificity

Specificity between primary and secondary antibodies in immunofluorescence experiments - practical applications

Specificity between primary and secondary antibodies in immunofluorescence experiments - practical applications

- Antibody binding is often dependent on the target protein maintaining its native conformational state.

- Access to a desired epitope can be compromised by interactions with other proteins, post-translational modifications, temperature, pH, fixation, and salt concentration.

- Because polyclonal antibodies are heterogeneous and can recognize multiple epitopes, they are less likely to be affected by changes in protein conformation.

- In general, polyclonal antibodies are also more stable than monoclonal antibodies over a range of pH and salt concentration.

- For these reasons, polyclonal antibodies are more frequently used for IHC/ICC experiments than monoclonal antibodies.

Specificity between primary and secondary antibodies in immunofluorescence experiments - practical applications

Careful selection and validation of primary antibodies: - Specificity is critical! - Must be validated for immunofluorescence - Must recognize fixed epitopes

Guidelines for validation: - Choose a primary antibody that is validated for immunofluorescence - Antibody must recognize fixed protein epitopes - Confirm antibody specificity to target proteins by Western blot - Confirm up- or down-regulation of target protein expression under various conditions - Use a blocking peptide to pre-adbsorb the primary antibody and reduce specific signal - Titrate antibodies for optimal signal and lowest background When purchasing primary antibodies from commercial suppliers, look for antibodies that are: - Validated for immunofluorescence applications - Affinity purified

Optimization of Primary Antibody Incubation - Primary antibody concentration, diluent, incubation time, and temperature all impact the

quality of staining. - These variables need to be optimized for each antibody and sample to achieve specific

staining and low background. - Often optimization is approached by maintaining a constant incubation time and

temperature, while varying the antibody concentration to determine when an optimal signal is achieved with low background noise.

- For example, if a high affinity antibody is used, then the antibody can be used at a relatively high concentration for a shorter incubation time.

- Alternatively, a high affinity antibody at lower antibody concentration may be used with a longer incubation time.

- Longer incubation durations are often employed to ensure penetration throughout tissue sections used for stereological techniques.

- To promote specific staining, longer incubation periods are often conducted at lower temperatures (i.e. 4 °C versus room temperature).

Antibody Validation By Western Blotting • Western blots yield useful information about specificity, even though antigen presentation is

different than in fixed cells • Confirm correct molecular weights of target protein

Arabidopsis thaliana whole leaf extract was separated by SDS-PAGE and blotted with: (Lane A) Rabbit Anti-AtMPK3 (0.5 μg/mL); (Lane B) Rabbit Anti-AtMPK4 (0.5 μg/mL); (Lane C) Rabbit Anti-AtMPK6 (0.25 μg/mL). The antibodies were developed with Goat Anti-Rabbit IgG-Peroxidase and a chemiluminescent substrate.

Arabidopsis thaliana whole leaf extracts (Lane A) and seedlings (Lane B), and tobacco whole leaf extract (Lanes C, D) were separated on SDS-PAGE, blotted with Rabbit Anti-AtMPK3 and developed using Goat Anti-Rabbit IgG-Peroxidase and a chemiluminescent substrate. Antibody concentration 1.0 μg/mL (Lanes A, B, C, E), 0.2 μg/mL (Lane D). Inhibition of antibody staining of Arabidopsis thaliana whole leaf extract with the AtMPK3 immunizing peptide (20 μg/mL, Lane E).

NOTE: - Some primary antibodies may not recognize denatured antigens on Western blots - Western blot specificity does not guarantee specificity in fixed cells - An antibody with strong non-specific bands may not be suitable for immunofluorescence

assays - Confirm that desired cellular response is triggered in your experiments - Optimize cell treatment conditions and timing to capture the peak response - Some physiological responses of cells before fixation, like phosphorylation levels, may

increase and decrease rapidly. Sampling at the wrong time may overlook or underestimate the response

Antibody Validation By Western Blotting

Combined Detection of Total Protein and Phospho-Protein Levels It may be possible to use the target protein as its own internal control for detection of a phospho-protein. Total levels of the target protein, and extent of phosphorylation of the target, are measured simultaneously. Combine the phospho-antibody with a "total protein" antibody that recognizes the target protein regardless of its phosphorylation status. For example, use mouse anti-total Akt and rabbit anti-phospho-Akt.

Combined Detection of Total Protein and Phospho-Protein Levels It may be possible to use the target protein as its own internal control for detection of a phospho-protein. Total levels of the target protein, and extent of phosphorylation of the target, are measured simultaneously. Combine the phospho-antibody with a "total protein" antibody that recognizes the target protein regardless of its phosphorylation status. For example, use mouse anti-total Akt and rabbit anti-phospho-Akt.

Specificity of secondary antibodies - Secondary antibodies are generated by immunizing a host animal with the antibody(s) from a

different species. - For example, anti-mouse antibodies are raised by injecting mouse antibodies into an animal

other than a mouse. Goat, donkey and rabbit are the most commonly used host species for raising secondary antibodies, but others may be available from individual suppliers.

- The most common types of secondary antibodies are those generated against a pooled

population of immunoglobulins from a target species. - For example, immunizing a goat with purified mouse IgG will generate goat anti-mouse IgG

antibodies that will bind to all classes, heavy and light chains (H&L) and fragments of mouse IgG as well as any other molecules sharing the same conserved domains (e.g., IgM share the same kappa light chains as IgG).

- In contrast, immunizing a goat with only mouse IgG1 antibodies will only generate antibodies

specific for mouse IgG1 antibodies and molecules sharing the same conserved domains.

Choosing a secondary antibody - In particular methods, typical secondary antibodies are either too specific (e.g., recognize

only one host species of primary antibody) or too general (e.g., recognize whole IgG and any fragments thereof).

- In most cases, these limitations can be overcome by carefully designing the experimental system and choosing the appropriate secondary probe.

The following considerations are useful to help choose a secondary antibody: - Determine the host species of the primary antibody (mouse anti-tubulin, rabbit anti-CD4,

etc.) - Select an appropriate host species for the secondary antibody (goat anti-mouse IgG, donkey

anti-rabbit IgG) - The choice of secondary antibody depends upon the species of animal in which the primary

antibody was raised (the host species). For example, if the primary antibody is a mouse monoclonal antibody then the secondary antibody must be an anti-mouse antibody obtained from a host other than the mouse.

Choosing a secondary antibody - In particular methods, typical secondary antibodies are either too specific (e.g., recognize

only one host species of primary antibody) or too general (e.g., recognize whole IgG and any fragments thereof).

- In most cases, these limitations can be overcome by carefully designing the experimental system and choosing the appropriate secondary probe.

The following considerations are useful to help choose a secondary antibody: - Consider cross-reactivity or specificity issues of the secondary antibody:

• Highly cross-absorbed – for multiple-labeling applications or when using samples with endogenous antibodies

• Specificity – binds to correct fragments, classes or chains of the primary antibody - Detection or purification method

• Label – appropriately conjugated to the correct enzyme, tag or fluorophore for the chosen detection method

• Ability to bind to Protein A, Protein G or Protein L – make sure the secondary antibody chosen has sufficient affinity for the molecules used upstream or downstream (i.e., Protein A-coated microplates.)

- Consider requirements of the supplied secondary antibody: • Supplied state – sterile liquid or lyophilized, suspended in PBS or Tris buffer, contains

carrier proteins such as gelatin or albumin or the addition of stabilizers such as sucrose or microbial inhibitors

• Because the vast majority of primary antibodies are produced in just a few host animal species, with nearly all of the antibodies being of the IgG class, it is easy and economical for manufacturers to produce and supply ready-to-use secondary antibodies that are applicable for most methods and detection systems.

• The secondary antibody must have specificity for the antibody species and isotype of the primary antibody being used.

• Given secondary antibody can be used with any primary antibody of the same type and host species, making it an infinitely more versatile reagent than individual labeled primary antibodies.

• From a relatively small number of secondary antibodies, many options are available for purity level, specificity and label type required for a given application.

• Secondary antibodies with specificity for the primary antibodies of common species are commercially available pre-conjugated with many of the common labels, making these detection reagents commodities.

Cross Reactivity

Specificity of secondary antibodies - Because of the high degree of conservation in the structure of many immunoglobulin

domains, class-specific secondary antibodies must be affinity purified and cross-adsorbed to achieve minimal cross-reaction with other immunoglobulins.

- Immobilized mouse IgG1 antibodies would be used to affinity purify all goat antibodies that

bind to mouse IgG1. These anti-mouse IgG1 antibodies would then be further purified by passage through a chromatography column(s) containing mouse IgG2a, IgG2b, IgG3, IgM, etc., to remove any antibodies that cross-react with non-IgG1 isotypes.

- Additionally, secondary antibodies can be further purified by passage through columns

containing the immobilized serum proteins from species other than those used to immunize the host.

- This method of cross-adsorption (often referred to as "Highly Cross-Adsorbed") is an

additional purification step recommended for applications where primary antibodies from multiple species will be used and when immunoglobulins or other serum proteins may be present in the samples being probed.

Pre-adsorbed secondary antibodies - Pre-adsorbed secondary antibodies are ideal for multi-color imaging and

immunohistochemistry experiments when several primary antibodies and their corresponding secondary antibodies are used simultaneously.

- Pre-adsorption (or cross-adsorption) is an extra step to increase the specificity of an antibody.

Pre-adsorbed secondary antibodies

Pre-adsorption process: Step 1. Antibodies, such as the mixture of antibodies recognizing rabbit IgG light-chains (A,B,C) shown in the above diagram, are passed through a matrix containing immobilized serum proteins. These proteins are from potentially cross-reactive species; in the above drawing, pre-adsorption against sheep and bovine was required and therefore serum proteins recognizing sheep and bovine light-chains were bound to the matrix. Step 2. Only antibodies specific to rabbit IgG light-chains (A) will pass through the column. Antibodies cross-reacting with sheep or bovine IgG light-chains (B and C) will bind and stay adsorbed to the matrix.

A B C

When using two antibodies against the same target, antibody interference may occur (one antibody blocks binding of the other antibody). Controls to rule out antibody inference are very important To simultaneously detect two target proteins in the immunofluorescence assay, you must: - Use two primary antibodies that differ in either host species or subclass/isotype - Use secondary antibodies labeled with different fluorophores

Blocking - Your staining will be improved by blocking the non-specific binding of your antibody to your

specimen. - BSA, milk or serum can be used for this. Don't use serum from the same species as the one

in which the primary antibody was raised! - If you use a secondary antibody developed from goat, a typical blocking solution is 5%

normal goat serum made in PBS-0.05% Tween-20. NaN3 can be added to 0.05% (w/v) to the solution. Blocking can last from 10 minutes to 30 minutes. Normally 50 µl-100 µl

- The most important part of the process - if it is good, you will get a bright, specific staining. - The perfect primary antibody has good affinity (ie it binds strongly to your protein of interest)

and good specificity (ie it doesn't bind to much else). - The concentrations of the antigen are much lower in IF than on an immunoblot, so the

antibody concentrations used are generally higher. - You will probably need to try a range of dilutions unless you have a protocol that you know

works well. - The best dilution for an antibody ranges from 1:10 to 1:10 000 dependent on the abundance

of the antigen, purity of your antibody, affinity between the antigen and the antibody, etc. - Dilute your antibody in the blocking solution. Double/triple staining The primary antibodies can be mixed as long as they came from different species of animals (most often mouse and rabbit).

(A) - Incubation with antibodies should show antibody binding to only the correct antigen. - Nonspecific binding can result from charged groups

that bind proteins, including antibodies. - The tissue can have Fc receptors that bind to the Fc

region of any antibody. (B) - There are blocking agents that block each of the sites

that cause nonspecific binding. - The primary antibody binds to the correct antigen

and is not affected by any of the blocking agents. - Charged groups can be quenched by any protein, and

BSA is commonly used because it is not a source of antibody binding.

- The Fc receptors must be quenched by the Fc end of an IgG antibody that has no ability to bind other antigens; normal serum from the same species as the secondary antibody is commonly used.

Blocking

Classical double immunolabeling experiment – two antibodies from two different hosts

Multicolor staining - The protocol for a double-staining procedure is highly dependent on the primary antibody

combination with respect to animal species, Ig isotype, mouse IgG subclass, or direct labeling.

- Investigators usually perform single staining first and then select the best antibody combination(s) for double staining.

- This strategy means that a double-staining protocol needs to be composed of primary antibodies that have previously been optimized for single staining.

- Consequently, a double-staining procedure has to be designed based on the characteristics of the primary antibodies involved.

- The aim of many double-staining experiments is colocalization: visualizing two markers in one

cell or tissue constituent. - Overlapping staining patterns result in a mixed-color when co-localization is found at the

same sub-cellular level (e.g. membrane, cytoplasm, and nucleus).

Multicolor staining In order to be able to examine the co-distribution of two (or more) different antigens in the same sample, a double immunofluorescence procedure can be carried out. This can be performed either simultaneously (in a mixture) or sequentially (one antigen after another).

Simultaneous incubation - Incubate cells with 1% BSA in PBST for 30 min to block unspecific binding of the antibodies

(alternative blocking solutions are 1% gelatin or 10% serum from the species that the secondary antibody was raised in).

- Incubate cells in the mixture of two primary antibodies (e.g. rabbit against human target-1 and mouse against human target-2, if the targets are human proteins) in 1% BSA in PBST in a humidified chamber for 1 hr at room temperature or overnight at 4°C.

- Decant the mixture solution and wash the cells three times in PBS, 5 min each wash. - Incubate cells with the mixture of two secondary antibodies which are raised in different

species (with two different fluorochromes, i.e. Texas Red-conjugated against rabbit and FITC-conjugated against mouse) in 1% BSA for 1 hr at room temperature in the dark.

- Decant the mixture of the secondary antibody solution and wash three times with PBS for 5 min each in the dark.

Sequential incubation - First blocking step: incubate cells with the first serum (10% serum from the species that the

secondary antibody was raised in) for 30 min to block unspecific binding of the antibodies at room temperature.

- Incubate cells with the first primary antibody in 1% BSA or 1% serum in PBST in a humidified chamber for 1 hr at room temperature or overnight at 4°C.

- Decant the first primary antibody solution and wash the cells three times in PBS, 5 min each wash.

- Incubate cells with first secondary antibody (labelled with Fluorochrome-1) in 1% BSA in PBST for 1 hr at room temperature in the dark.

- Decant the first secondary antibody solution and wash three times with PBS for 5 min each in the dark.

- Second blocking step: incubate cells with the second serum (10% serum from the species that the secondary antibody was raised in) for 30 min to block unspecific binding of the antibodies at room temperature in the dark.

- Incubate cells with the second primary antibody in 1% BSA in PBST in a humidified chamber in the dark for 1 hr at room temperature or overnight at 4°C depending on the concentration of the antibody and the accessibility of the antigen.

- Decant the second primary antibody solution and wash the cells three times in PBS, 5 min each wash in the dark.

- Incubate cells with second secondary antibody (labelled with Fluorochrome-2) in 1% BSA for 1 hr at room temperature in the dark.

- Decant the second secondary antibody solution and wash three times with PBS for 5 min each in the dark.

Immunofluorescence Double Staining Protocol - Sequential Approach for Primary Antibodies Raised from Same Species

1. Rinse Sections in PBS-Tween 20 for 2x2 min.

2. 1st Serum Blocking: incubate sections in normal serum – species same as secondary antibody (for example:

1st primary antibody is mouse and 1st secondary antibody is horse anti-mouse, so horse normal serum

block should be used).

3. 1st Primary Antibody: incubate sections in 1st primary antibody (mouse) at appropriate dilution in antibody

dilution buffer for 1 hour at room temperature. Notes: (1) do not rinse between step 2 and 3; (2) room

temperature means 22-25 °C. If too low, longer incubation time is needed; (3) some antibodies may require

overnight incubation.

4. Rinse in PBS-Tween 20 for 3x2 min.

5.1st Secondary Antibody: incubate sections in FITC conjugated horse anti-mouse secondary antibody in PBS

for 20-30 minutes at room temperature.

6.Rinse in PBS-Tween 20 for 3x2 min.

7.2nd Serum Blocking: use normal mouse serum to block free binding sites of the anti-mouse immunoglobulin.

8.2nd Primary Antibody: incubate sections in 2nd primary antibody (mouse) at appropriate dilution in antibody

dilution buffer for 1 hour at room temperature.

9.Rinse in PBS-Tween 20 for 3x2 min.

10.2nd Secondary Antibody: incubate sections in Texas Red conjugated anti-mouse secondary antibody in PBS

for 20-30 minutes at room temperature.

11.Rinse in PBS-Tween 20 for 3x2 min.

12. Counterstain with DAPI if desired for 20 minutes at room temperature.

13. Rinse in PBS-Tween 20 for 3x2 min.

14.Coverslip with anti-fade mounting medium.

15.Store slides in dark at 4 °C.

Immunofluorescence Double Staining Protocol - Parallel Approach

1. Rinse Sections in PBS-Tween 20 for 2x2 min.

2.Serum Blocking: incubate sections in normal serum blocking solution – species same as secondary antibody

(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).

3.Primary Antibodies: incubate sections in the mixture of two primary antibodies (mouse and rabbit) at

appropriate dilution in antibody dilution buffer for 1 hour at room temperature.

4.Rinse in PBS-Tween 20 for 3x2 min.

5.Secondary Antibodies: incubate sections in the mixture of two fluorescent conjugated secondary

antibodies (FITC conjugated Horse anti-Mouse and Texas Red conjugated Goat anti-Rabbit) in PBS for 30

minutes at room temperature).

6. Rinse in PBS-Tween 20 for 3x2 min.

7.Counterstain with DAPI if desired for 20 minutes at room temperature.

8. Rinse in PBS-Tween 20 for 3x2 min.

9.Coverslip with anti-fade fluorescent mounting medium and seal with nail polish.

10. Store slides in dark at 4 °C.

Multicolor staining - The mouse-rabbit combination is the easiest double staining procedure to perform

technically. - A cocktail of both primary antibodies and then both secondary antibodies followed by the

subsequent visualization of AP activity and HRP activity makes this indirect-indirect simultaneous double-staining protocol a short and user-friendly procedure.

- Because both anti-mouse, anti-rabbit polymers contain a secondary antibody of goat origin, there is no interspecies cross-reaction problem.

mouse-rabbit indirect-indirect simultaneous combination for double staining red-brown

Multicolor staining - Mouse-mouse combinations - Because many antibodies are of murine monoclonal origin, it is obvious that a mouse-mouse

combination is regularly needed. - Indirect/indirect simultaneous double-staining protocol based on two mouse monoclonal primary antibodies of different Ig type (IgG-IgM) or different IgG subclass (IgG1, IgG2a, IgG2b, IgG3)

mouse-mouse indirect-indirect simultaneous double staining in red-brown, based on lgG subclass differences of the primary antibodies

Multicolor staining - Mouse-mouse combinations - Because many antibodies are of murine monoclonal origin, it is obvious that a mouse-mouse

combination is regularly needed. - Indirect/indirect simultaneous double-staining protocol based on two mouse monoclonal primary antibodies of different Ig type (IgG-IgM) or different IgG subclass (IgG1, IgG2a, IgG2b, IgG3)

mouse-mouse/FITC, indirect-direct multistep double staining in red-brown

Multicolor staining - In their simplest form, these techniques use primary antibodies that are directly labeled by

different fluorophores. - In this way, simultaneous detection of up to seven antigens has been described, provided

that properly selected dyes, suitably designed filter sets, and a sophisticated analysis are available.

- Although useful in certain applications, direct methods suffer from a lower sensitivity than indirect methods.

- Investigators use indirect two- or multi-layer methods which, given the availability of primary antibodies raised in different species, can be directly used for multiple stainings in combination with different fluorophore-labeled species-specific secondary antibodies.

- An elegant double staining method is the use of fluorochrome-conjugated monovalent Fab fragments as secondary antibodies.

- In this approach the secondary polyclonal monovalent Fab fragments used to visualize the first primary antibody prevent the secondary antibodies, used to detect the second primary antibodies, from binding to the first primary antibody.

Multicolor staining - triple immunofluorescence labeling method using three commercially available polyclonal antibodies raised in rabbit against neuronal nitric oxide synthase (nNOS), calcitonin gene-related peptide (CGRP), and calbindin D28k (CB) in the lung of rats. Biotin-conjugated tyramide signal amplification (TSA) enhancement for detection of the first antibody, fluorophore-labeled and unlabeled Fab secondary antibodies for the second antibody, and standard fluorophore-labeled secondary antibodies for the third primary antibody.

Co-localisation of Cx40, Cx43 and Cx45 in clone 6.(A) Each connexin can be seen in the individual channels

within the same area and the triple labelling image was generated by superimposing the image for each connexin

subtype. (B) To demonstrate the varying degrees of co-localisation at each cell interface, sections 1 and 2

highlighted from triple labelling were separated into dual images of Cx40/Cx43, Cx40/Cx45, Cx43/Cx45 and triple

image of Cx40/Cx43/Cx45 as indicated on the left panel of the image.

The primary antibodies used were our custom-designed guinea pig polyclonal anti-Cx45 ((Q14E) GP42) [5], a

rabbit polyclonal anti-Cx40 ((S15C) R83) [5] and a mouse monoclonal anti-Cx43 (Chemicon MAB-3068). For

single labelling appropriate secondary antibodies labelled with Cy3 were used. For the triple labelling

Cx40/Cx43/Cx45, the anti-Cx45 was applied first, followed by the anti-Cx43 and then the anti-Cx40. For the

secondary antibodies the anti-guinea pig labelled with Cy3 was applied first, then the anti-mouse labelled with

FITC followed by the anti-rabbit labelled with Cy5.

Multicolor staining Schematic diagram of the optimized general protocol for double and multiple antigen labeling in IHC, with the first primary antibodies raised in the same species and of the same species IgG isotype.

Multicolor staining

Superclonal™ Secondary Antibodies

- antibody designed to eliminate cross-reactivity and background in secondary antibody detection. - recombinant antibody technology designed to provide precise and accurate detection of mouse, rabbit and goat primary antibodies in a variety of applications. Features of Superclonal secondary antibodies: - Designed to eliminate cross-reactivity in detection of primary antibodies - Developed as recombinant monoclonal antibodies to enable precise and accurate detection - Formulated to recognize both heavy- and light-chain epitopes (H+L) of target IgG molecules - Selected and optimized for use with cell imaging, ELISA, and western blotting applications - Offered in four types: goat anti-mouse (GAM), goat anti-rabbit (GAR), rabbit anti-mouse

(RAM), rabbit anti-goat (RAG) - Available unconjugated and conjugated with biotin, horseradish peroxidase (HRP), and

selected Alexa Fluor™ dyes

Performance of Superclonal™ secondary antibodies. The nucleoli of HeLa cells were labeled with anti-nucleostemin primary antibody, which was then detected with respective types of Invitrogen™ Alexa Fluor™ 488 conjugated secondary antibodies (green): (A) highly cross-adsorbed goat anti-mouse IgG (H+L) secondary antibody, or (B) Goat Anti-Mouse IgG (H+L) Superclonal Secondary Antibody. Cell nuclei were stained with DAPI (blue) and actin filaments were stained with Alexa Fluor 594 Phalloidin (red). Detection with the Superclonal secondary antibody exhibits significantly less cytoplasmic staining, indicating enhanced specificity.

Superclonal™ Secondary Antibodies