Technical Tips: Immunohistochemical Staining

1. Troubleshooting Guide for Immunohistochemistry
2. Cultured Cells or Cytospins
3. Paraffin Sections
4. Frozen Sections
5. General Instructions

1. Troubleshooting Guide for Immunohistochemistry   Top

No brown staining	No counterstaining or counterstaining is too light
Brown staining too light	Counterstain is too dark
Sections are too brown or entire section is brown	Staining pattern is not what is expected
Debris found on slides

 

Problem: No "brown" staining
Possible causes:
•	Were the primary and secondary antibodies added?
•	Concentration of primary antibody may be too low. Increase the concentration by a factor of 2 - 3.
•	Make sure the H2O2 was added to the DAB solution. Use a fresh stock bottle of H2O2.
•	Was the enyzme pretreatment step performed?
•	Slides left too short a time in the DAB solution.
•	Is the tissue type known to express the antigen?
•	Pretreatment was too long and the epitope was destroyed. Reduce the incubation time.
•	Were the antibodies stored at the correct temperature? Antibodies sold in solution will be inactivated by freezing, while antibodies sold lyophilized are best stored frozen after they are reconstituted.
•	Is the tissue of the correct species origin?
Problem: "Brown" staining too light
Possible causes:
•	Primary antibody concentration too low. Increase the concentration by a factor of 2 - 3.
•	Increase the incubation time with the primary antibody.
•	Slides left too short a time in the DAB solution.
•	Level of expression may be low for the specific tissue sample.
•	How old are the reagents/kit? Check expiration date on vial(s).
•	Enhance staining by placing slides in 0.5% cupric sulfate in 0.15 M NaCl for 5 minutes. Rinse in de-ionized H2O, then counterstain with hematoxylin.
Problem: Sections are too brown or entire section is brown
Possible causes:
•	Primary antibody concentration too high. Decrease the concentration by a factor of 2 - 3.
•	Slides left in the DAB solution too long. Shorten the incubation time in DAB.
•	Tissue was not properly blocked. Increase the incubation time with the blocking serum and/or increase the concentration of blocking serum by a factor of 3. If BSA is used, be sure it is fatty acid-free.
•	Tissue was not properly blocked. Increase the incubation time with the blocking serum and/or increase the concentration of blocking serum by a factor of 3. If BSA is used, be sure it is fatty acid-free.
•	Washing steps in between incubations with antibody were shortened or omitted altogether.
•	Preincubation with H2O2 blocking solution was not performed.
•	H2O2 is old. New reagent should be obtained.
•	Cross-reactivity between the secondary antibody and proteins in the tissue may be occurring. Add one or both:
	- 1% normal rabbit serum (Calbiochem Cat. No. NS01L) to the blocking solution.
	- 0.01% TRITON® X-100 (Calbiochem Cat. No. 648462) to the primary antibody solution (caution: this may also reduce specific binding of some antibodies).
Problem: Debris found on the slides
Possible causes:
•	DAB is old and beginning to precipitate out of solution. Filter DAB solution before using.
•	Tissue sections are of poor quality. New sections should be obtained.
•	Xylene is dirty and should be changed.
Problem: No counterstaining or counterstaining is too light
Possible causes:
•	Increase time left in the Hematoxylin solution.
•	Use a "bluing" reagent to enhance the staining.
Problem: Counterstain is too dark (no contrast is seen between the counterstain and DAB)
Possible causes:
•	Sections left in hematoxylin solution too long. Reduce the incubation period.
•	Insufficient incubation with DAB. Increase the incubation time in DAB.
•	Try an alternate histological counterstain such as methyl green.
•	Place sections in 70% ethanol with 1% HCl (1N) until stain lightens.
Problem:Staining pattern not what is expected (cytoplasmic as well as nuclear)
Possible causes:
•	Tissue was over digested with the enzyme pretreatment step. Reduce the duration of the incubation or the concentration of enzyme used.
•	Sections were improperly blocked with the serum blocker. Increase incubation time for the blocking step or increase the concentration of blocker used.
•	Excess primary antibody was used. Decrease the concentration of primary antibody. Cross-reactivity between the secondary antibody and proteins in the tissue may be occurring. Add 1% normal rabbit serum to the blocking solution or reduce the amount of secondary antibody by a factor of 3.
2. Cultured Cells or Cytospins®   Top
Overview:
Calbiochem monoclonal and polyclonal antibodies can be used for localization of antigens in fixed cultured cells. Note: Cytospins® of cells can be formalin-fixed and paraffin- embedded, then pretreated and stained as with paraffin sections above.
Required Solutions and Reagents:
•	Cells: on cover slips
•	PBS (phosphate buffered saline): dissolve 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4, and 0.24 g of KH2PO4 in 800 mL of distilled water. Adjust the pH to 7.4 with HCl. Add distilled water to 1 liter.
•	Fixatives as desired: 1% formalin, 100% methanol, or 100% acetone.
Procedure: Preparation of Slides
•	Grow cultured cells on sterile glass cover slips or slides (such as chamber slides) overnight at 37°C.
•	Rinse briefly with PBS.
•	Fix as desired. Possible procedures include (use only one):
•	- 10 minutes with 1% formalin in PBS (keep wet). - 5 minutes with -10°C methanol. Allow to air dry. - 5 minutes with cold acetone. Allow to air dry.
Immunoperoxidase Staining
	Stain using a commercially available kit, such as Calbiochem UniTect™ kits (Cat Nos. XHC01, XHC02), following the manufacturers instructions.
3. Paraffin Sections   Top
Overview:
Calbiochem monoclonal and polyclonal antibodies which are recommended for paraffin sections can be used for localization of antigens in formalin-fixed, paraffin-embedded tissue section.
Solutions and Reagents:
•	Tissue sections: formalin- fixed, paraffin-embedded sections (4 to 8 microns thick)
•	0.1% H2O2 in distilled water - prepare fresh daily.
•	Distilled water
•	PBS (phosphate buffered saline): dissolve 8 g of NaCl, 200 mg of KCl, 1.44 g of Na2HPO4, and 240 mg of KH2PO4 in 800 mL of distilled water. Adjust the pH to 7.4 with HCl. Add distilled water to 1 liter.
•	Pretreatment reagents as needed (see Pretreatment of Tissue Sections below)
•	Solvents: 100% and 95% ethanol; xylene
Procedure: Deparaffinization of Formalin-fixed, Paraffin-embedded Tissue Sections
1.	Deparaffinize in xylene, using 3 changes, 5 minutes each.
2.	Hydrate with 100% ethanol, using 2 changes, 5 minutes each.
3.	Hydrate with 95% ethanol, using 2 changes, 5 minutes each.
4.	Rinse in distilled water.
5.	Incubate for 30 minutes in 0.1% H2O2 to quench any endogenous peroxidase activity.
6.	Rinse in distilled water.
7.	Rinse in PBS, using 3 changes, 5 minutes each.
8.	Follow procedure for pretreatment as required.
Pretreatment of Paraffin-Embedded Tissue Sections:
Antigenic determinants masked by formalin-fixation and paraffin-embedding may be exposed by enzymatic digestion, saponin or heat. Do not use with frozen sections or cells which are not paraffin-embedded. The different treatments are summarized below:
•	Pronase® Protease : incubate sections for 4 to 6 minutes in 0.0025% PRONASE® Protease (Calbiochem Cat. No. 53702) in 10 mM Tris buffer, pH 7.6, at room temperature. Terminate digestion by washing in PBS containing 0.2% glycine.
•	Trypsin: incubate sections for 12 minutes in 0.1% Trypsin (Calbiochem Cat. No. 6502) in PBS at room temperature.
•	Terminate digestion by incubation with 0.1 mg/mL Soybean Trypsin Inhibitor (Calbiochem Cat. No. 65035) in PBS for 5 minutes.
•	Pepsin: incubate sections for 10 - 20 minutes in 0.1% Pepsin (Calbiochem Cat. No. 516360) in 0.01 N HCl, pH 2.3, at room temperature. Terminate by repeated washing in distilled water.
•	Saponin: incubate sections for 30 minutes in 0.05% saponin, (Calbiochem Cat. No. 558255) in distilled water at room temperature. Wash at least three times in PBS.
•	Neuraminidase: incubate sections for 60 minutes in 0.02 units/mL Neuraminidase, (Calbiochem Cat. No. 480717) in PBS at room temperature. Wash at least three times in PBS.
•	Heat: incubate for 10 minutes at 95°C in 10 mM sodium citrate (pH 6.0) buffer. Allow slides to cool to room temperature before addition of primary antibody and continuing with staining procedure. Note: superior results may be obtained with a pressure cooker (see below).
•	4 N HCl: incubate sections for 10 minutes in 4 N HCl at 37°C. Wash at least three times in distilled water.
Pressure Cooker Pretreatment of Formalin-fixed, Paraffin-embedded Sections:
Expose antigenic sites and reduce background by heat treatment of formalin-fixed, paraffin-embedded tissue sections in a citrate buffer using pressure cooker pretreatment for 10 minutes.
*Wear eye protection and thermal gloves for steps 4 and 5.
1.	Place slide holder in plastic coplin jar containing 200 mL of 10 mM sodium citrate buffer, pH 6.0. Transfer slides to be tested to slide holder. Loosely place cover on coplin jar, leaving room for steam to escape.
2.	Add 1 liter of water to a pressure cooker containing a steaming rack. Place pressure cooker on a laboratory hot plate. Place the coplin jar(s) in the pressure cooker (up to 4 jars can be processed simultaneously). Attach the lid securely to the pressure cooker and then place the pressure counter weight on top of the vent in the center of the lid.
3.	Turn the heat setting on the hot plate to high. After about 30 minutes the center weight on the pressure cooker lid will begin to rock as steam escapes. Reduce heat setting on hot plate to midrange. Set timer for 10 minutes.
4.	When 10 minutes has elapsed, turn the hot plate off and carefully transfer the pressure cooker to a heat-resistant surface.
5.	After 10 minutes cooling, remove the counter weight to allow steam to escape. When the safety lock has dropped back into position, the lid may be opened and the coplin jars removed.
6.	Remove slides and allow to cool for an additional 15 minutes. Transfer slides to PBS solution for 5 minutes.
7.	Proceed with standard protocol for IHC staining.
Immunoperoxidase Staining
Stain using a commercially available kit, such as Calbiochem UniTect™ kits (Cat. Nos. XHC01, XHC02), following the manufacturers instructions.
4. Frozen Sections   Top
Overview:
	Calbiochem monoclonal and polyclonal antibodies recommended for frozen sections can be used for localization of antigens in frozen tissue sections.
Required Solutions and Reagents:
•	Tissue sections: cryostat cut frozen sections (4 to 8 microns thick)
•	Subbing Solution: 0.3% (w/v) gelatin, 0.05% chromium potassium sulfate in distilled water (if using uncoated glass slides)
Procedure: Preparation of Frozen Sections
1.	Clean glass slides with 95% ethanol, treat with subbing solution (or use coated slides), and air dry.
2.	Cut 4 to 8 micron thick cryostat sections of tissue block (frozen in isopentane precooled in liquid nitrogen, embedded in OCT compound in cryomolds, and stored at -70°C).
3.	Allow frozen sections to come to room temperature (30 minutes).
4.	Fix slides in cold acetone for 10 minutes, keep refrigerated (or choose another fixation procedure).
5.	Rinse in three changes of PBS.
6.	Incubate for 5 to 10 minutes in 0.1% hydrogen peroxide (H2O2) in PBS to quench endogenous peroxidase activity.
7.	Rinse in PBS.
Immunoperoxidase Staining
Stain using a commercially available kit, such as Calbiochem UniTect™ kits (Cat. Nos. XHC01, XHC02), following the manufacturer's instructions.
5. General Instructions   Top
Overview
Calbiochem monoclonal and polyclonal antibodies recommended for immunohistochemistry can be used for localization of antigens in tissue sections or cultured cells by immunoperoxidase staining. A method employing the streptavidin-biotin system is recommended. For convenience, appropriate second step reagents for immunohistochemistry are available from Calbiochem, as complete staining kits (UniTect kits, Cat. Nos. XHC01, XHC02). In the immunoperoxidase staining procedure, target cells or tissue sections, either frozen or formalin-fixed and paraffin-embedded, are first incubated with a monoclonal or polyclonal antibody to the antigen of interest. Specifically bound antibody is then visualized by incubation with a biotinylated second-step antibody against immunoglobulins of the relevant species (e.g. biotinylated goat anti-mouse IgG), followed by incubation with a streptavidin-horseradish peroxidase conjugate and substrate. Using diaminobenzidine as substrate, positive staining is observed as a brown precipitate. This has been termed the ABC technique and is currently the most sensitive immunoperoxidase method available. The avidin D-biotinylated horseradish peroxidase H complex consists of many biotinylated horseradish peroxidase molecules cross-linked by avidin to form a three dimensional array. The complex apparently has few exposed biotin residues but retains at least one biotin binding site. Formation of the complex is achieved by mixing defined amounts of avidin DH and biotinylated horseradish peroxidase H in dilute solution prior to use. This complex remains stable for several hours after formation.
Equipment:
•	Coplin (or equivalent) staining jars
•	Beakers
•	Hanging slide racks
•	Humidifying chamber
•	Coverslips
•	Light microscope
Required Solutions and Reagents:
•	Monoclonal or polyclonal primary antibody
•	Detection kit or reagents, such as biotinylated goat anti-mouse, anti-rat or anti-rabbit IgG and streptavidin-horseradish peroxidase
•	PBS (phosphate buffered saline): dissolve 8 g of NaCl, 200 mg of KCl, 1.44 g of Na2HPO4,and 240 mg of KH2PO4 in 800 mL of distilled water. Adjust the pH to 7.4 with HCl. Add distilled water to 1 liter.
•	PBS-BSA: PBS with 1% (w/v) Fatty Acid Free Bovine Serum Albumin (Calbiochem Cat. No. 126575)
•	10% (v/v) Normal Goat Serum (Calbiochem Cat. No. NS02L) in PBS
•	Triton-PBS: 1% TRITON® X-100 (Calbiochem Cat. No. 648462) in PBS
•	DAB (diaminobenzidine tetrahydrochloride, 3-3′ diaminobenzidine, Calbiochem Cat. No. 281751): dilute as per product instructions (or dissolve 5 mg DAB in 100 mL of PBS and add 0.1 mL of 0.3% hydrogen peroxide). Prepare fresh DAB solution daily. Caution: use DAB in a fume hood-it is a suspected carcinogen.
•	Aqueous Mounting Medium
•	Hematoxylin (Harris type)
•	Solvents: ethanol and xylene
Procedure: Immunoperoxidase Staining
•	Carry out incubations at room temperature or at 4°C in a humidified chamber. Use suction to remove reagents after each step, but avoid drying of specimens between steps. Use sufficient reagent to cover the specimen (usually 20 to 50 mL).
•	Incubate specimens with 10% Normal Goat Serum (Calbiochem Cat. No. NS02L) for 20 minutes to suppress non-specific binding of immunoglobulin.
•	Wash with PBS, using 3 changes, 15 minutes each.
•	Incubate with primary antibody in PBS-1% BSA for 60 minutes at room temperature, or overnight at 4°C. Overnight incubation is recommended for formalin-fixed, paraffin-embedded sections. Optimal antibody concentration for a given application should be determined by titration; a starting concentration is given in the data sheet.
•	Rinse with three changes of PBS, using 3 changes, 5 minutes each.
•	Incubate with appropriate biotin conjugated second step antibody in PBS for 45 minutes. Optimal antibody concentration for a given application should be determined by titration; a starting concentration is given in the data sheet.
•	Rinse with three changes of PBS, using 3 changes, 5 minutes each.
•	Incubate with Streptavidin-Horseradish Peroxidase (Cat No. OR03L) in PBS for 15 minutes. Streptavidin conjugate concentration for a given application should be determined by titration; a recommended starting concentration is given in the data sheet.
•	Wash extensively with PBS.
•	Rinse in TRITON®-PBS for 30 seconds.
•	Incubate in DAB solution for 5 minutes. Monitor under microscope. If further intensification of staining is required, return to DAB and incubate for additional 1 to 5 minutes.
•	Rinse in distilled water.
•	Counterstain in hematoxylin solution for 1 - 3 minutes if desired.
•	Dehydrate through alcohols and xylene.
•	Observe by light microscopy.

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