United States Biological

Key Features

Stringent Quality Control Testing

DNase, RNase, protease, bioburden, growth performance, strain selection assays, blue-white cloning, background fluorescence, restriction enzyme digestion, endotoxin, 9CFR sterility testing, physical and chemical properties.
Quality Control and Manufacturing Guidelines followed include IACUC, NIH, GLP and GMP.

New Express 35 Day Antibody Development

With United States Biological you do not have to wait for 80 days until the rabbit gives a good response. Their new technology speeds up the development of the immune response; in 35 days the animals give titers of specific antibodies (IgG’s) which can be even higher than after 60-80 days of a standard (multiple injection) immunisation scheme.

Molecular Biology Services

Current services available to customers include polyclonal and monoclonal antibody production, cDNA and genomic library construction, library screening, custom cloning and expression, large-scale plasmid purification, protein expression, peptide synthesis and ELISA development.

Antibodies

All Antibodies

View All Epitope Tags

Epitope Tags

The use of epitope tags eliminates the need to generate a new antibody for each protein saving time, resources and money.

View All Matched Pairs

Matched Pairs

Capable of detecting different epitopes on the same protein antigen, Monoclonal pairs, polyclonal antibody pairs, and hundreds of antibody-conjugate pairs have been validated in sandwich assays such as ELISA and lateral flow.

View Custom Antibodies

Custom Antibodies

Antibodies are produced in a USDA certified animal research facility, and comply with the Animal Welfare Act. A typical standard protocol includes animal purchase, housing and care, all immunizations, bleeds per our standard protocol formats.

View All Primary Antibodies

Primary Antibodies

Choose from qualified antibodies for Immunology, Cancer, Epigenetics, etc… validated and guaranteed.

View All Secondary Antibodies

Secondary Antibodies

Available in many formats and are useful in a wide range of applications, including Flow Cytometry, Immunocytochemistry and Western Blotting.

View All Isotype Controls

Isotype Controls

Isotype controls are non-reactive immunoglobulins used as negative controls to help differentiate non-specific background signal from specific antibody signal.

Antibody Protocols

The protocols described below are for general application. Any product specific protocol supercedes these general recommendations.

Immunofluorescence

Background

Immunofluorescence is a technique that allows the visualization of a specific protein or antigen in cells or tissue sections by binding a specific antibody chemically conjugated with a fluorescent dye such as fluorescein isothiocyanate (FITC). There are two major types of immunofluorescence staining methods: 1) direct immunofluorescence staining in which the primary antibody is labeled with fluorescence dye, and 2) indirect immunofluorescence staining in which a secondary antibody labeled with fluorochrome is used to recognize a primary antibody. Immunofluorescence staining can be performed on cells fixed on slides and tissue sections. Immunofluorescence stained samples are examined under a fluorescence microscope or confocal microscope.

Because fluorescent dyes such as fluorescein and rhodamine can be coupled to antibodies without destroying their specificity, the conjugates can complex with antigen and be visualized via fluorescence microscopy. The microscope excites the chosen dyes by light of one or more wavelengths, which in turn emits light at a characteristic wavelength captured by a selective filter. The image is then projected with localized regions of fluorescence indicating different antigens labeled by antibodies of distinctive color.

Common Fluorescent Tags: Excitation & Emission

Common Fluorescent Tags	Excitation (nm)	Emission (nm)
Fluorecein	495	530
Fluorescein isothiocyanate	492	518
B-Phycoerythrin	546	575
R-Phycoerythrin	490	575
Texas Red	568	590
Rhodamine	555	620
Allophyycocyanin	650	660

Direct Immunofluorescence

The antibody is itself conjugated with the fluorochrome and applied directly to a monolayer of cells or to frozen tissue on a slide. When examined with a fluorescence microscope, the antibody labeled with the fluorescent compound identifies the localized antigen.

Indirect Immunofluorescence

Unlike direct immunofluorescence, indirect immunofluoresence is a double-layer technique. The unlabeled antibody is applied directly to the tissue substrate and then treated with a fluorochrome-conjugated anti-IgG. There are several advantages to this technique, and it is typically used more frequently than the direct method. Because several fluorescent anti-immunoglobulins can bind to each antibody present in the first layer, this produces brighter fluorescence than in the direct method. It is also more time-efficient since there is only one fluorescent-labeled reagent, the anti-IgG prepared during the lengthy conjugation process. A general protocol for only the indirect immunofluorescence technique is presented here.

Immunofluorescence Protocol – Tissues

Materials required
Tissue Slides
Coverslips
Slide racks & tray
Staining dishes with lids
Orbital Shaker
PAP pen & Transfer pipettes
Deionized water (ddH2O)
PBS (Phosphate Buffered Saline)
Primary antibody
Fluorescent-labeled secondary antibody
Normal Serum
Glycerol

Sample Preparation

Samples are perfused or dissected and fixed in 4% paraformaldehyde fixative.
Transfer the tissue to 20% sucrose in PBS, leave overnight at 4°C.
Transfer the tissue to 30% sucrose in PBS, leave at 4°C to impregnate fully. When the tissue sinks, it is fully impregnated.
Freeze and cut 5-20mm thick crysostat sections

Blocking Step

Incubate slides for 1 hour in 5% normal serum (serum of secondary antibody host) in PBS.
Wash with PBS, 3x 5 minutes.

Primary Antibody Incubation

Incubate with fluorescence-conjugated secondary antibody in PBS, 0.1% normal serum for 1-2 hours at RT in the dark. (From this point on, keep slides in the dark.)
Wash 3x with PBS. To reduce background can include 0.1% Tween 20 in PBS.

Fluorescence Detection

Mount coverslip.
Examine using fluorescence microscopy immediately or store flat at 4°C in the dark.

Immunofluorescence Protocol – Cells

Note: Cells should be grown, treated, fixed, and stained directly in multiwell plates, chamber slides, or on coverslips.

Materials required

PBS
Paraformaldehyde
Methanol
Distilled water (dh2O)
PBST: 1X PBS, 0.1% Triton X-100. To prepare 1L, add 100ml 10X PBS to 900ml dH2O. Add 1ml
Triton X-100 and mix.
Primary antibody
Fluorochrome-conjugated secondary antibody
Mounting Medium

Specimen Preparation

To increase cell adherence, treat coverslips with a 1:10 dilution of poly-lysine solution at room temperature for 5 minutes.
Plate cells at appropriate dilution and grow until cells reach desired confluence (~70%)
Aspirate media
Rinse cells briefly in PBS.

Fixation Step

Aspirate PBS, cover cells with 2-4% formaldehyde in PBS (work in fume hood). Allow cells to fix for 15 minutes at RT.
Aspirate fixative, rinse 3x in PBS for 5 minutes each.

Permeabilization (optional)

Methanol and acetone Fixation result in permeabilized cell preparations. For paraformaldehyde fixed preparations, treat with 0.2% Triton X-100 for 5 min or alternatively with -20°C methanol for 5 minutes.

Methanol Permeabilization Step: After formaldehyde fixation, cover cells with ice-cold 100% methanol (use enough to cover cells completely to a depth of 3-5mm, do not let cells dry), incubate cells in methanol for 10 minutes in freezer.
Rinse in PBS for 5 minutes.

Blocking Step

Note: All incubations should be carried out at RT unless otherwise noted in a humid light-tight box or covered dish to prevent drying and to prevent exposure of fluorochrome to light.

Block sample in 5% normal serum from same species as secondary antibody (eg. normal goat serum, normal donkey serum) in PBS,1%Triton X-100 for 1 hour. (Recommended Blocking Buffers can vary between 1-2% Bovine Serum Albumin, Fetal Bovine Serum Albumin, or 2% Non-fat dry milk in TBS (PBS) with or without 0.2% Tween 20 and 0.02% Sodium Azide.)

Primary Antibody Incubation

Dilute primary antibody in PBS, 0.1% Triton. Typical volumes are: 50-100ul per section, 25-50ul per coverslip, chamber, or well (48 or 96 well plate).
Aspirate blocking solution, apply diluted primary antibody.
Incubate overnight at 4°C with gentle agitation or rocking.
Rinse 3x in PBS, 0.1% Triton X-100 for 5 minutes each.

Secondary Antibody Incubation

Incubate with fluorochrome-conjugated secondary antibody diluted in PBS, 0.1% Triton X-100 for 1-2 hours at RT in dark. (From this point on, slides need to be kept in the dark.)
Rinse in PBS, 0.1% Triton X-100.

Fluorescence Detection

Mount coverslips.
Examine using fluorescence microscopy immediately or store flat at 4°C in the dark.

Immunoprecipitation

Background

Immunoprecipitation is a method that enables the purification of a protein. Additionally, one can use immunoprecipitation to confirm the identity or study biochemical characteristics, post-translational modifications, and expression levels of a protein of interest. An antibody for the protein of interest is incubated with a cell extract so that the antibody will bind the protein in solution. The antibody/antigen complex will then be pulled out of the sample using protein A/G-coupled agarose beads. This physically isolates the protein of interest from the rest of the sample. The sample can then be separated by SDS-PAGE for Western blot analysis.

Immunoprecipitation Protocol

Lysis Buffers

The ideal lysis buffer will leave proteins in their native conformation, minimizing denaturation of antibody binding sites while at the same time releasing adequate amounts of protein from the sample for subsequent analysis. Other variables that can affect the success of IP include salt concentration, divalent cation concentration, and pH.

NP-40 Cell Lysis Buffer: 50mM Tris-HCl pH 8.0, 150mM NaCl, 1% NP-40
Protease Inhibitor Cocktail (100X): PMSF, 5mg (50ug/ml), Aprotinin, 100ug (1μg/ml), Leupeptin, 100ug (1ug/ml), Pepstatin, 100ug (1ug/ml), 100% Ethanol bring up to 1ml, aliquot and keep at -20°C. (Add to Lysis buffer just before use.)
RIPA (RadioImmunoPrecipitation Assay) buffer (alternate) RIPA buffer is particularly useful for nuclear membrane disruption for nuclear extracts. RIPA buffer gives low background but can denature kinases. 50mM Tris HCl, pH 8, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS (10% sodium deoxycholate stock solution (5g into 50ml) must be protected from light.)

Preparation of Lysates

Harvest approximately 10e7 cells. Note: The total number of cells/ml and the cell equivalent loaded per lane of gel should be optimized specifically for each protein and antibody.
Wash cells with ~10 ml of PBS in a conical tube and spin at 400xg for 10 minutes. Discard supernatant.
Wash cells with ~10 ml of PBS in a conical tube and spin at 400xg for 10 minutes.
After the second wash, remove supernatant completely and resuspend the cell pellet in 1ml of cold Lysis Buffer containing 1X Protease Inhibitor Cocktail (final concentration of 10e7 cells/ml). Gently vortex the tube. Note: To effectively prevent protease action, the Lysis Buffer should be pre-chilled and all the following steps should performed at 4°C. For adherent monolayers, add 1ml of cold Lysis Buffer containing 1X Protease Inhibitor Cocktail per 100mm culture dish.
Place the tube or the dish on ice for 30 minutes, with occasional mixing. Spin cell lysate at 10,000xg for 15 minutes at 4°C.
Carefully collect supernatant, without disturbing the pellet, and transfer to a clean tube. The cell lysate can be frozen at this point for long-term storage at -70°C. Discard the pellet.

Pre-clearing the lysates

If using a monoclonal antibody choose protein G-coupled Sepharose beads; if using a polyclonal antibody, protein A-coupled Sepharose beads are usually suitable (refer to Table 1).

Transfer 50ul of the Protein G beads slurry to an eppendorf tube and add 450ul cold Lysis Buffer. Spin at 10,000xg for 30 seconds and remove the Lysis Buffer.
Wash one more time with 500ul of cold Lysis Buffer. Resuspend the beads in 50ul of cold Lysis Buffer.
Add the 50ul of prepared Protein G slurry and 500ul of Cell Lysate to an eppendorf tube and incubate on ice for 30-60 minutes.
Spin at 10,000xg for 10 minutes at 4°C and transfer the supernatant to a fresh eppendorf. If any bead has been transferred, spin again and carefully transfer the supernatant to another fresh eppendorf tube.

Immunoprecipitation

Add 5-10ug of antibody to the eppendorf tube containing the cold precleared lysate. Note: This concentration of monoclonal antibody is suggested as a starting point. Incubate at 4°C for 1 hour.
Add 50ul of washed Protein G slurry in prechilled Lysis Buffer (prepared as instructed in Preclearing Step 1 above).
Incubate for 1 hour at 4°C on a rocking platform or a rotator.
Spin the eppendorf tube at 10,000xg for 30 seconds at 4°C.
Carefully remove the supernatant completely and wash the beads 3-5 times with 500ul of Lysis Buffer. To minimize background, care should be given to remove the supernatant completely in these washes.
After the last wash, aspirate supernatant and add 50ul of 1X Laemmli sample buffer to bead pellet.
Vortex and heat to 90-100°C for 10 minutes.
Spin at 10,000xg for 5 minutes, collect supernatant and load onto the gel. Supernatant samples can be collected and kept frozen at this point if the gel is to be run later.

Table 1: Choosing the Correct Beads-Summary

Species	Immunoglobulin Isotype	Protein A	Protein G
Human	IgG1	+++	+++
Human	IgG2	+++	+++
Human	IgG3	–	+++
Human	IgG4	+++	+++
Human	IgM	Use anti Human IgM	Use anti Human IgM
Human	IgE	–	+
Human	IgA	–	+
Mouse	IgG1	+	+++
Mouse	IgG2a	+++	+++
Mouse	IgG2b	++	++
Mouse	IgG3	+	+
Mouse	IgM	Use anti Mouse IgM	Use anti Mouse IgM
Rat	IgG1	–	+
Rat	IgG2a	–	+++
Rat	IgG2b	–	++
Rat	IgG2c	+	++
Chicken	All isotypes	–	++
Cow	All isotypes	++	+++
Goat	All isotypes	–	++
Guinea Pig	All isotypes	+++	++
Hamster	All isotypes	+	++
Horse	All isotypes	++	+++
Pig	All isotypes	+	++
Rabbit	All isotypes	+++	++
Sheep	All isotypes	–	++

Immunocytochemistry

Background

Immunocytochemistry is an immunological technique that is very similar to Immunohistochemistry. Immunocytochemistry is used to visualize the presence of a specific protein or antigen in cells (cultured cells, cell suspensions), rather than tissues. The types of cell samples that can be investigated include blood smears, cultured cells, cell suspensions, and cytospins. Each type of cell sample is prepared and treated slightly differently, but the fundamental use of primary antibody, secondary antibody and color development is very similar between Immunocytochemistry and Immunohistochemistry.

For immunocytochemistry, sample preparation involves fixing the target cells to a slide. Cells can be attached to a solid surface by several methods: adherent cells may be grown on microscope slides; cell suspensions can be centrifuged onto glass slides (cytospin),or bound to solid support using chemical linkers.

To ensure access of the antibody to its antigen, cells must be fixed and permeabilized. In an ideal situation, fixation would immobilize the antigens while retaining native cellular architecture and permitting unhindered access of antibodies to all cells and subcellular compartments.

Fixation methods fall generally into two groups: organic solvents and cross-linking reagents. Organic solvents, such as alcohols and acetone, remove lipids and dehydrate the cells, while precipitating the proteins on the cellular architecture. Cross-linking reagents (such as paraformaldehyde) form intermolecular bridges, normally through free amino groups, thus creating a network of linked antigens. Cross-linkers preserve cell structure better than organic solvents, but may alter the structure of some cell components, so much so that they are not recognized by the primary antibody.

Fixation Methods

Acetone Fixation Fix cells in -20°C acetone for 5-10 minutes. No permeabilization step is needed following acetone fixation.
Methanol Fixation Fix cells in -20°C methanol for 5-10 minutes. No permeabilization step needed following methanol fixation.
Ethanol Fixation Fix cells in cooled 95% ethanol, 5% glacial acetic acid for 5-10 minutes.
Methanol-Acetone Fixation Fix cells in cooled methanol, 10 minutes at -20°C. Remove excess methanol. Permeabilize with cooled acetone for 1 minute at –20°C.
Methanol-Acetone Fixation Prepare 1:1 methanol and acetone mixture and fix cells at -20°C for 5-10 minutes.
Methanol-Ethanol Fixation Prepare 1:1 methanol and ethanol mixture and fix cells at -20°C for 5-10 minutes.
Formalin Fixation Fix cells in 10% neutral buffered formalin for 5-10 minutes. Rinse briefly with PBS. Permeabilize with 0.5% Triton X-100 for 10 minutes.
Paraformaldehyde-Triton Fixation Fix cells in 3-4% paraformaldehyde for 10-20 minutes. Rinse briefly with PBS. Permeabilize with 0.5% Triton X-100 for 10 minutes.
Paraformaldehyde-Methanol Fixation Fix cells in 4% paraformaldehyde for 10-20 minutes. Rinse briefly with PBS. Permeabilize with cooled methanol for 5-10 minutes at -20°C.

Permeabilization Methods

Cross-linkers typically require the addition of a permeabilization step to allow access of the antibody to the specimen. Two groups of compounds can be used for permeabilization: organic solvents and mild surfactants. Alcohols and acetone can be used as a one step fixation/permeabilization method. In addition to their fixative behavior which precipitates proteins in place, these solvents also dissolve membrane lipids which render the membrane permeable to antibodies. In the surfactant category, saponin, Triton X-100, Tween-20, etc, are used for their ability to disrupt membranes.

Immunocytochemistry (HRP detection)

Materials Required

Deionized water
PBS
Triton X-100
Hydrogen Peroxide (H2O2)
Primary Antibody
Biotinylated secondary antibody (or HRP-conjugated secondary antibody)
Bovine Serum Albumin (BSA)
Streptavidin-HRP
DAB
Glycerol
Hematoxylin (optional)
Acetic Acid (optional)

Sample Preparation

Transfer cell culture to the wells of a chamber slide or slides of your choice. Allow cells to grow to confluence with the addition of fresh media.
Wash the cells thoroughly 5×2 min in PBS.

Fixation Step

Fix cells with appropriate fixation method (see above).
Rinse 5×2 min in PBS.

Permeabilization Step

Incubate the slides with 0.25-0.5% Triton X-100 (or other permeabilization medium) in PBS for 10 minutes to permeabilize the membranes (Note: there is no need for a permeabilization step following acetone or methanol fixation).
Rinse 3×5 min in PBS.

Endogenous Peroxidase Blocking Step

Block endogenous peroxidase by incubating in 3% H2O2 in PBS for 10-30 minutes (This step is required only if an HRP conjugated secondary antibody is to be used.)
Rinse in 3x5min in PBS.

Blocking of Non-specific Binding

Block with 2-5% normal serum in PBS for 1 hour. Normal serum should be the same species from which the secondary antibody was raised. (Alternatively, 5% BSA is sometimes used as blocking agent.)

Primary Antibody Incubation

Dilute the primary antibody to the recommended concentration in 1% normal serum, PBS.
Remove blocking buffer from the slides.
Add primary antibody to each well. Incubate for 1-2 hours at RT or overnight at 4°C.
Remove the primary antibody solution and rinse slides 3 x 5 minutes in PBS.

Secondary Antibody Incubation

Dilute the biotinylated secondary antibody in 1% BSA diluent.
Remove the excess fluid from the slide and add secondary antibody solution into each well. Incubate for 1 hour at RT.
Rinse 3 x 5 minutes in PBS. Remove excess fluid.

Color Development

Add streptavidin-HRP to each well. Incubate for 30 minutes at RT.
Wash 3 x 5 minutes in PBS on an orbital shaker. Remove excess fluid.
Add DAB solution to each cell well. Once the cells start turning brown (this can be observed under a microscope) wash 2 x 5 minutes in PBS on the shaker.

Optional Counterstain

Dip the slides into a staining dish of hematoxylin for 30 seconds.
Remove and place into an acid bath (200ml dH2O and 1-3 drops of acetic acid). Rinse with dH2O.

Cover Slips

Add cover slips to the slides for examination under the microscope

Immunohistochemistry

Background

Immunohistochemistry is widely used in basic research to understand the distribution and localization of biomarkers and differentially expressed proteins in different parts of a biological tissue. IHC staining is also widely used in the diagnosis of abnormal cells such as those found in cancerous tumors. Visualizing an antibody-antigen interaction can be accomplished in a number of ways. In the most common instance, an antibody is conjugated to an enzyme or cofactor, such as biotin, horseradish peroxidase (HRP) or alkaline phosphatase (AP) that can catalyze a color-producing reaction. Alternatively, the antibody can also be tagged to a fluorophore. such as fluorescein (FITC) or R-Phycoerythrin (PE) (see Immunofluorescence).

With the use of a conjugated primary antibody, direct IHC can be performed, but this method is seldom used because of the amplifying effect available with the use of a conjugated secondary antibody. The choice of secondary antibody for Immunohistochemistry is similar to that of ELISA and Western Blot. The conjugated secondary is directed specifically at the host species of the primary antibody and its specific isotype (e.g., mouse IgG1, goat IgM, rabbit IgG1,k, chicken IgY, etc.). In addition, if working with tissues or cells that have Fc receptors (thymus, spleen, blood, hematopoietic cells, leukocytes, B cells, etc.), it is best to choose an F(ab’)2 fragment to eliminate non-specific binding through Fc receptors present on cells. As an alternative, binding of IgG whole molecule secondary antibodies to Fc receptors may be blocked by incubating cells in purified normal serum from the host species of the secondary antibody.

Immunohistochemistry (Fresh Frozen Sections)

Materials Required:

Acetone
Ethanol, anhydrous denatured, histological grade (100% and 95%)
dH2O
Hematotoxylin
Xylene
10X TBS: To prepare 1 liter of 10X TBS: 24.2g Tris base, 80g sodium chloride; adjust pH to 7.6 with HCl (use at 1X).
Wash Buffer TBS/T: 1X TBS, 0.1% Tween-20: To prepare 1 liter add 100ml of 10x TBS to 900ml ddH2O. Add 1ml Tween 20 and mix.
10mM Sodium Citrate Buffer: To prepare 1 liter, add 2.94g sodium citrate to 1 liter dH2O. Adjust pH to 6.0.
3% Hydrogen Peroxide: To prepare, add 10ml 30% H2O2 to 90ml dH2O.
Blocking Solution: 5% horse serum or goat serum in TBS
ABC Reagent: Prepare according to manufacturer’s instructions 30 minutes before use.
DAB Reagent: Prepare according to manufacturer’s recommendations.

Tissue Preparation:

Snap-freeze small tissue blocks (5x5x3mm) in liquid nitrogen.
Transfer to cryostat, cut into thin (5–30um) sections, and transfer sections to positively charged slides (poly-L-lysine coated).
Dry the slides at RT (or 1-2 hours until completely dry if performing staining on the same day). Note: Thorough drying is required for proper adhesion to the slides.

Fixation methods:

A variety of fixation methods are available. Follow the specific methods mentioned in the product datasheet, or find the optimal method for your sample:
Cold acetone: 10 minutes at -20°C. Air dry
Methanol: 10 minutes at -20°C
10% Neutral buffered formalin: 10 minutes at RT
3% Formaldehyde: 15 minutes at RT
3% Formaldehyde/methanol: 15 minutes at RT, followed by 5 minutes in methanol at -20°C (do not rinse in between)
Wash slides 3 times, 5 min each in PBS, pH 7.4 containing 1% Tween 20.

Fixed, Frozen Tissue Sections

Perfuse tissue with fixative or immerse tissue in fixative for a set time period. 4% paraformaldehyde is the most commonly used fixative.
Immerse the tissue in cyroprotectant solution containing 10-30% sucrose in PBS. Cryoprotection is complete when the tissue no longer floats in the solution.
Remove tissue from the cyroprotectant solution and store at -70°C until sectioned.
Remove tissue from the -70°C freezer and equilibrate at -20°C for about 15 minutes before attempting to section. Equilibration helps prevent cracking of the block when sectioning.
Using a cryostat, prepare 10-15um sections that can be collected directly onto slides. Usually 3 sections can be placed per slide; spaced well apart.
Thoroughly dry sections on slides. Drying can be accomplished by air drying or by using a slide warmer, usually overnight, or at least 2-3 hours at 40-50°C.
Prepared slides can be stored dry at -70°C until stained. Equilibrate to room temperature and briefly re-dry prior to rehydration and staining.

Paraffin-Embedded Sections

Deparaffinize and hydrate sections:
Xylene 2-3 changes, 5 minutes each
100% absolute ethanol: 2 changes, 3 minutes each
95% ethanol: 2 changes, 3 minutes each
80% ethanol: 3 minutes
50% ethanol: 3 minutes
Distilled water, PBS, or Tris buffer: 2 changes, 3 minutes each.

Note: Once the tissue sections have been rehydrated, do not allow them to dry. Dry the slide around the tissue section with an absorbent wipe. Using a diamond pencil, china marking pencil or fingernail polish, draw a circle on the microscope slide around the section. This circle will help retain solution on the section during subsequent incubations with reagents.

Perform antigen retrieval if required. (For antigen retrieval methods see Section V. HIER (heat-induced epitope retrieval) using citrate buffer is a commonly used method.
Block endogenous peroxidase (if required) by immersing slides into 0.3-3% H2O2 and 100% methanol for 10-30 minutes at RT.
Wash sections in distilled water, 2 changes for 5 minutes each.

Immunostaining

Blocking

Incubate sections with 3-10% normal serum from the same species as the secondary antibody, for 30 minutes to block non-specific binding of immunoglobulin.
Remove blocking solution.

Primary Antibody Incubation

(All steps should be performed in a moist environment.)

Dilute the primary antibody in blocking solution. If no dilution is suggested, begin testing at 1:10, 1:100 and 1:1000.
Incubation overnight at 4°C.
Remove antibody solution. Wash 3 x 5 minutes in PBS, pH 7.4.
If the primary antibody is HRP-conjugated, proceed to Color Development.

Secondary Antibody Incubation

Dilute the biotin-conjugated secondary antibody in blocking solution according to the recommended dilution. Incubate 30-60 minutes at RT.
Removed secondary antibody solution. Wash slides 3 x 5 minutes in wash buffer.

Color Development

Add streptavidin-horseradish peroxidase reagent to each section and incubate for 30 min. at RT.
Remove ABC reagent. Wash sections 3x in wash buffer for 5 min. each.
Apply the DAB solution to cover the sections completely in a moist environment. Incubate for 5-15 minutes at RT. Alternatively, observe the slide under a microscope to determine optimal color intensity of the insoluble precipitate.
As soon as color develops, stop the reaction by gently flushing with dH2O.
Counterstain tissue as desired with Hematoxylin & Eosin to define antigen proximity to normally expected structures.
Wash the slides with dH2O.

Dehydration and coverslip application

Dehydrate the sample for storage using a series of methanol or ethanol graded concentrations: 50% (2x5min), 75% (2x5min), and finally 100% (2x5min).
Repeat in xylene, incubating sections two times for 10 seconds each.
Allow the slide to air dry.
Mount coverslips

Antigen Retrieval

The visualization of many antigens can be significantly improved by pretreatment with antigen retrieval methods that break the protein cross-links formed by formalin fixation and thereby uncover hidden antigenic sites. Antigen retrieval techniques typically involve either the application of heat for varying lengths of time or the use enzymatic digestion by using proteases such as proteinase K, trypsin, or pepsin.

The heating methods typically utilize either a microwave oven, pressure cooker, steamer or water bath. Samples are heated for 20 minutes at close to 100°C, followed by cooling for an equivalent length of time. The most frequently used retrieval solutions are a) citrate buffer, pH 6.0, b) Tris-EDTA, pH 9.0 and c) EDTA, pH 8.0.

Antigen Retrieval using Citrate Buffer
Citrate buffer: 10mM sodium citrate, 0.05% Tween-20, pH 6.0 or 10mM citric acid, 0.05% Tween-20, pH 6.0

Pre-heat steamer or water bath with staining dish containing Sodium Citrate Buffer or Citrate Buffer until temperature reaches 95-100°C.
Immerse slides in the staining dish. Place the lid loosely on the staining dish and incubate for 20-40 minutes (optimal incubation time should be determined by user).
Turn off steamer or water bath and remove the staining dish to room temperature and allow the slides to cool for 20 minutes.
Rinse sections in TBS-Tween-20 for 2×2 min.
Proceed to blocking step.

Troubleshooting

Problem: Weak or no staining

Antibodies do not work due to improper storage: Aliquot antibodies into smaller volumes and store in freezer (-20°C) and avoid repeated freeze/thaw cycles; store antibodies according to manufacturer’s instructions.
Antibody concentration was too low: Increase the concentration of primary and/or secondary antibodies. Or run a serial dilution test to determine the optimal dilution that gives the best signal to noise ratio.
Inadequate antibody incubation time: Increase antibody incubation time.
Inadequate or improper tissue fixation: Increase duration of postfixation or try different fixatives.
Incompatible secondary and primary antibodies: Use secondary antibody that will interact with primary antibody. For example, if primary antibodies are raised from rabbits, use anti-rabbit secondary antibodies.
Inactive secondary antibody: Replace with a new batch of antibody.
Inactive streptavidin-horseradish peroxidase reagent: Replace with a new batch of reagents.
Defective or incompatible enzyme substrate (DAB) system: Replace with a new batch of reagents.
Inadequate substrate incubation time: Increase the substrate incubation time.

Problem: High Background

Inadequate washing of sections. Wash at least 3x between steps.
Tissue contains endogenous enzyme such as peroxidase or alkaline phosphatase. Block endogenous enzyme activities using 3% hydrogen peroxide (blocks peroxidase) in methanol or levamisole (blocks AP) prior to incubation with primary antibody
Tissue contains endogenous biotin activity. Block endogenous biotin activity using the avidin-biotin blocking reagent prior to incubation with primary antibody.
Mouse antibody used on mouse tissues. Treat tissue with mouse on mouse blocking reagent prior to the primary antibody incubation.
Sections dried out. Avoid letting sections dry out.

Flow Cytometry

Background

Staining of cells using monoclonal antibodies is used extensively to characterize cell surface antigens when monitoring extracellular antigens. Flow Cytometry data have been used to identify the families of cell types based on surface antigens and to group antibodies based on their recognition of these antigens. The use of flow cytometry for extracellular staining continues to provide useful data for identifying and separating specific cell populations. The method requires specialized equipment in which a mixture of cells can be separated based on the presence or absence of the expected emission wavelength of a specific fluorescent conjugate.

Common Examples of Fluorescent Dyes: Peak Wavelength (nm)

Fluorochrome	Abbreviation	Absorption	Emission
Fluorecein	FITC	495	530
Rhodamine	Rhod	555	620
R-Phycoerythrin	rPE	490, 546	575
Texas Red	TR	568	590
R-Cy5	Cy5	488	680

Flow Cytometry Protocol for Extracellular Antigens

Note: Fluorescent reagents should be protected from light.

Materials and Equipment
Sample cells
12 x 75mm test tubes
PBS, 0.1% sodium azide, 1% FBS
Fluorescent-labeled Antibody
Centrifuge
Vortex mixer
Flow cytometer
PBS, 0.5% Paraformaldehyde, 4°C
Pipettes
PBS/ sodium azide at 4°C

Procedure

Prepare the desired biological cells according to the appropriate protocol. Adjust the concentration of the cells to 2x10e7 cells per ml (or 1x10e6 cells per 50ul: each test consists of 1x10e6 cells), diluting with PBS, 0.1% sodium azide, 1% FBS or BSA, as necessary. The cells can be isolated up to 4 hours before being stained as long as they are kept on ice. Note: In some cases, use of a non-specific binding or blocking agent may be desired. If so, add the blocking agent (such as normal serum or BSA) and incubate at RT for 10 minutes prior to the addition of antibody to the cells.
Dilute the fluorescent conjugated antibody appropriately according to the specific recommended dilution for the staining being done. Use PBS, 0.1% sodium azide, 1% FBS for the dilution. Dilute the antibody so that a volume of 10-20ul is added to the cells.
Incubate the cells with the antibody at 4°C for 30 minutes in the dark. All conjugated antibodies (FITC, R-PE, etc.) for double or triple staining can be added simultaneously at this point and do not require additional incubations.
After incubation remove the unbound antibody from the cells by washing with 1ml of PBS, 0.1% sodium azide, 1% FBS. Pipette the PBS, 0.1% sodium azide, 1% FBS into each tube, vortex, then centrifuge at 350xg for 5-7 minutes at 4°C. Carefully aspirate the supernatant leaving about 50-100ul in the bottom of each tube. Repeat this process for a total of 3 washes.
If flow cytometry is to be performed the same day, resuspend the cells in 0.5ml of cold PBS, 0.1% sodium azide, 1% FBS after aspirating the supernatant. Gently vortex and analyze the cells. If analysis will not be performed the same day, the cells may be fixed in 0.5ml cold PBS with 0.5% paraformaldehyde and stored at 2-8°C in the dark in buffer containing 0.1% sodium azide for as long as 48 hours.

Flow Cytometry for Intracellular Antigens
Cell Fixation and Permeabilization

Note: Cells should be kept on ice unless otherwise specified

Wash the cells twice with cold PBS.
Fix the cells with 2% paraformaldehyde in cold PBS for 15 minutes (4°C). It is very important to assure that the cells are uniformly resuspended during fixation.
Wash the cells twice with cold PBS.
Permeabilize the cells with 100% ice-cold methanol added dropwise while the cells are gently vortexing. Again, it is vitally important that the cells are uniformly suspended. Allow the cells to soak in cold methanol for 15 minutes. (Alternate permeabilization methods include the use of 0.1% saponin or 0.1% Tween-20.)
Wash the cells twice with cold PBS.

Antibody Incubation

Note: Use isotype matched controls for monoclonal antibodies or species matched IgG for polyclonal antibodies. Count cells using a hemocytometer or alternative method.

Add approximately 1x10e6 cells to each flow cytometry tube and wash with 1 ml of 0.1% saponin or Tween 20 diluted in PBS with 2% FBS added.
Resuspend cells in 1 ml of 0.1% saponin (or Tween 20) + 2% FBS and incubate for 30 minutes at RT. The purpose of this step is to block non-specific binding.
Add 20ul of antibody diluted to the recommended concentration in 100 ul of the above blocking solution and incubate for 20 minutes at RT.
If the primary antibody is not conjugated with a fluorochrome, then a second incubation with a fluorochrome-conjugated secondary antibody will be necessary. Incubate 20 minutes in blocking solution at RT.
Wash the cells once with blocking buffer, then finally with PBS
Resuspend the cells in 500ul PBS and run on flow cytometer.

Troubleshooting

Problem: No staining

Make sure that the appropriate primary or secondary antibodies have been added, and that the secondary antibody has the correct host and source to correctly bind to the primary antibody.
Is the secondary antibody active? Has it been used successfully with other primary antibodies?
If the fluorochrome used is Phycoerythrin or Allophycocyanin based, make sure that the label has not been frozen.
Is the target antigen present on test tissue? Check literature for antigen expression and incorporate a positive control of known antigen expression alongside test material.
Does antibody recognize antigen in test species? Check that antibody cross-reacts with species being used. Not all antibodies will cross-react across species.
Ensure that correct laser is being used to excite fluorochrome, and that correct channel is being used to analyze emissions.

Problem: Non-specific staining

Non-specific staining may be due to autofluorescence. Solution: check levels of autofluorescence by including a tube of cells only (i.e. without any antibody) into your panel.
Certain cells express low affinity Fc receptors, which bind whole antibodies via Fc region. Use Fc blocking agents.
Non-specific staining may be due to the secondary antibody. Select a secondary antibody that will not cross-react with target tissue.
Make sure that sufficient washing steps have been included.
Titrate the test antibody carefully. Non-specific staining may be reduced at lower antibody concentrations.

Problem: Weak staining

Weak staining may be due to overdilution of antibodies. Make sure that antibodies are used at the correct concentration by titrating antibodies before use.
Weak staining may be due to an excess cell number. Adjust cell population to recommended density.
Weak staining may be due to lack of antigen expression. Check literature for expected levels of expression.
If antigen expression is weak, select an antibody that is conjugated to a brighter fluorochrome.
Incubation time and temperature with either primary or secondary antibody should be optimized.

Problem: Unexpected staining profile

Ensure that cells are used as fresh as possible. Profile may be showing dead cells and debris.
Lysing solutions may affect certain antigens. Select a method that does not interfere with antigen detection.
Some antigens are expressed intracellularly and, therefore, cell permeabilization methods may be required.