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6: Immunoperoxidase Staining
A. Tissue Culture Cells
Grow cultured cells on sterile glass cover slips or slides (Cover Glasses and Micro-slides for Immunohistochemistry) overnight at 37º C. Wash briefly with PBS (Buffers and General Solutions) and fix cells by one of the following procedures:
1) 5 minutes in -10º C methanol, air dry (recommended method); or
2) 2 minutes in cold acetone, air dry; or
3) 10 minutes in 1% formalin in PBS (keep wet).
Wash in three changes of PBS.
Optional: Incubate for 5–10 minutes in 0.1–1% hydrogen peroxide in PBS to quench endogenous peroxidase activity. Wash in PBS twice for 5 minutes each.
B. Frozen Tissue Sections
Freeze tissue in block in liquid nitrogen according to standard procedures. Block may be stored at -70º C for up to 2 weeks before sectioning.
Clean glass slides with 95% ethanol, treat with subbing solution and air dry. Or use pre-treated slides.
Cut 4- to 10-micron thick sections. Adhere sections to room temperature slides. Slides may be stored at -70º C. Thaw slides at room temperature prior to fixing and staining.
NOTE: For a listing of mounting media for Immunohistochemistry including Organo/Limonene and UltraCruz™ Mounting, see Mounting Media for Immunohistochemistry.
Fix slides in cold acetone for 10 minutes and keep refrigerated (or choose other fixation procedure). Wash in three changes of PBS (Buffers and General Solutions).
Optional: Incubate for 5–10 minutes in 0.1–1% hydrogen peroxide in PBS to quench endogenous peroxidase activity. Wash in PBS twice for 5 minutes each.
NOTE: For tissues containing high levels of endogenous biotin (which may result in higher background staining), we recommend following the Formalin-Fixed, Paraffin-Embedded Tissue Sections protocol, as endogenous biotin is normally destroyed in paraffin-embedded tissue.
C. Formalin-Fixed, Paraffin-Embedded Tissue Sections
Fix tissue sections in formalin and embed in paraffin blocks according to standard procedures.
Clean glass slides with 95% ethanol, treat with subbing solution and air dry. Or use pre-treated slides.
Cut 4–6 micron thick tissue sections, and apply to slides. Deparaffinize in xylenes using three changes for 5 minutes each. Hydrate sections gradually through graded alcohols: wash in 100% ethanol twice for 10 minutes each, then 95% ethanol twice for 10 minutes each. Wash in deionized H2O for 1 minute with stirring. Aspirate excess liquid from slides.
Optional: Antigen unmasking may be performed at this point. Certain antigenic determinants are masked by formalin fixation and paraffin embedding and may be exposed by one of several methods:
1) Heat treatment (recommended method): Place slides in a container and cover with 10 mM sodium citrate buffer, pH 6.0; or with 50 mM glycine-HCl buffer (glycine: sc-29096), pH 3.5, with 0.01% (w/v) EDTA (EDTA: sc-29092). Heat at 95º C for 5 minutes. Top off with fresh buffer and heat at 95º C for 5 minutes (optimal incubation time may vary for each tissue type). Allow slides to cool in the buffer for approximately 20 minutes. Wash in deionized H2O three times for 2 minutes each. Aspirate excess liquid from slides.
2) Pepsin: Incubate sections for 10–20 minutes in 0.1% pepsin in 0.01 N HCl at room temperature. Wash slides several times in deionized H2O. Aspirate excess liquid from slides.
3) Saponin: Incubate sections for 30 minutes in 0.05% saponin in deionized H2O at room temperature. Wash at least three times in PBS. Aspirate excess liquid from slides.
Optional: Incubate for 5–10 minutes in 0.1–1% hydrogen peroxide in deionized H2O to quench endogenous peroxidase activity. Wash in PBS twice for 5 minutes each.
D. Immunoperoxidase Staining
For immunoperoxidase staining of tissue sections, we recommend the use of either the Santa Cruz Biotechnology, Inc. ImmunoCruz™ ABC or LSAB Staining Systems. The ImmunoCruz™ ABC Staining Systems utilize preformed avidin-biotinylated horseradish peroxidase complex as a detection reagent, whereas the ImmunoCruz™ LSAB Staining Systems utilize an Avidin D-HRP complex. The ImmunoCruz™ Staining Systems include all secondary reagents in a pre-diluted, ready to use format. Complete research protocols are included with all Staining Systems; brief protocols are given below.
All steps are carried out at room temperature in a humidified chamber. Allow all Staining System reagents to reach room temperature prior to use. Tissue sections should not be allowed to dry out at any time during the procedure. Use suction to remove reagents after each step, but avoid drying of specimens between steps. Use sufficient reagents to cover the specimens (approximately 100 µl per slide is usually adequate).
ImmunoCruz™ ABC Staining Systems
Incubate specimens for 1 hour in 1.5% normal blocking serum in PBS (Buffers and General Solutions). Blocking serum (Normal Sera for Immunohistochemistry) ideally should be derived from the same species in which the secondary antibody is raised. Remove blocking serum from slides.
Incubate with primary antibody for 30 minutes at room temperature or overnight at 4º C. Optimal antibody concentration should be determined by titration; recommended range is 0.5–5.0 µg/ml diluted in PBS with 1.5% normal blocking serum. Wash with three changes of PBS for 5 minutes each.
Incubate for 30 minutes with biotin-conjugated secondary antibody as provided, or at approximately 1 µg/ml diluted in PBS with 1.5% normal blocking serum. Wash with three changes of PBS for 5 minutes each.
Incubate for 30 minutes with avidin biotin enzyme reagent. Wash with three changes of PBS for 5 minutes each.
Incubate in peroxidase substrate as provided for 30 seconds–10 minutes, or until desired stain intensity develops. Individual slides should be monitored to determine the proper development time. Wash sections in deionized H2O for 5 minutes. If desired, counter-stain in Gill´s formulation #2 hematoxylin (sc-24973) for 5–10 seconds. Immediately wash with several changes of deionized H2O.
Dehydrate through alcohols and xylenes as follows: Soak in 95% ethanol twice for 10 seconds each, then 100% ethanol twice for 10 seconds each, then xylenes three times for 10 seconds each. Wipe off excess xylene. Immediately add 1–2 drops of permanent mounting medium (e.g., Clarion sc-24942), cover with a glass coverslip (sc-24975) and observe by light microscopy.
ImmunoCruz™ LSAB Staining Systems
Incubate specimens for 20 minutes in 1–3 drops of serum block. Aspirate serum from slides.
Dilute primary antibody in serum block to 0.5–5.0 µg/ml as determined by titration. Incubate for 2 hours. Rinse with PBS (Buffers and General Solutions) then wash in PBS twice for 2 minutes each on a stir plate. Aspirate excess liquid from slides.
Incubate for 30 minutes in 1–3 drops of biotinylated secondary antibody. Wash as above.
Incubate for 30 minutes in 1–3 drops of Avidin D-HRP complex. Wash as above.
Add 1–3 drops HRP substrate mixture. Develop for 30 seconds–10 minutes, or until desired stain intensity develops. Rinse with deionized H2O and transfer to a deionized H2O wash for 2 minutes on a stir plate.
Counterstain, dehydrate and mount slides as described under ABC Staining Systems.


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Immunoperoxidase Staining
ImmunoCruz™ ABC and LSAB Staining Systems

Isotype-Specific Secondary Antibodies for Immunohistochemistry

Control IgGs and IgG Conjugates

Secondary Antibodies for Immunohistochemistry

F(ab')2 Secondary Antibodies for Immunohistochemistry

Mounting Media for Immunohistochemistry

Normal Sera for Immunohistochemistry

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