NOTE: For a listing of offered cell culture products including classical and specialty media, sera and media additives, induction agents, antibiotics, attachment and dissociation agents, see General Laboratory Support Products.
1. Grow cultured cells on sterile glasscover slips or slides (Cover Glasses and Micro Slides forImmunohistochemistry) overnight at 37º C. Wash briefly with PBS(Buffers and General Solutions) and fix cells by one of thefollowing 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).
2. Wash in threechanges 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.
1. Freeze tissue in block in liquidnitrogen according to standard procedures. May be stored at -70º Cfor up to 2 weeks before sectioning.
2. Clean glass slides with 95% ethanol,treat with subbing solution and air dry. Or use pre-treatedslides.
3. 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 andstaining.
NOTE: For a listing of mounting media forImmunohistochemistry including Clarion and Crystal Mounting Media,see Mounting Media for Immunohistochemistry.
4. Fix slides in cold acetone for 10minutes 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 ofendogenous biotin (which may result in higher background staining),we recommend following the Formalin-Fixed, Paraffin-Embedded TissueSections protocol, as endogenous biotin is normally destroyed inparaffin-embedded tissue.
1. Fix tissue sectionsin formalin and embed in paraffin blocks according to standardprocedures.
2. Clean glass slides with 95% ethanol,treat with subbing solution and air dry. Or use pre-treatedslides.
3. Cut 4–6 micron thick tissue sections,and apply to slides. Deparaffinize in xylenes using three changesfor 5 minutes each. Hydrate sections gradually through gradedalcohols: wash in 100% ethanol twice for 10 minutes each, then 95%ethanol twice for 10 minutes each. Wash in deionizedH2O for 1minute with stirring. Aspirate excess liquid from slides.
Optional: Antigen unmasking may be performed atthis point. Certain antigenic determinants are masked by formalinfixation and paraffin embedding and may be exposed by one ofseveral methods:
1) Heat treatment (recommended method): Placeslides in a container and cover with 10 mM sodium citrate buffer(sodium citrate: sc-29109), pH 6.0; or with 50 mM glycine-HClbuffer (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 bufferand heat at 95º C for 5 minutes (optimal incubation time may varyfor each tissue type). Allow slides to cool in the buffer forapproximately 20 minutes. Wash in deionized H2O three timesfor 2 minutes each. Aspirate excess liquid from slides.
2) Pepsin: Incubate sections for 10–20 minutes in 0.1% pepsin in0.01 N HCl at room temperature. Wash slides several times indeionized H2O. Aspirateexcess liquid from slides.
3) Saponin: Incubate sections for 30 minutes in 0.05% saponin indeionized H2O at roomtemperature. Wash at least three times in PBS. Aspirate excessliquid from slides.
Optional: Incubate for 5–10 minutes in 0.1–1%hydrogen peroxide in deionized H2O toquench endogenous peroxidase activity. Wash in PBS twice for 5minutes each.
NOTE: For tissues containing high levels ofendogenous biotin (which may result in higher background staining),we recommend following the Formalin-Fixed, Paraffin-Embedded TissueSections protocol, as endogenous biotin is normally destroyed inparaffin-embedded tissue.
1. For immunoperoxidasestaining of tissue sections, we recommend the use of either theSanta Cruz Biotechnology, Inc. ABC Staining Systems or theImmunoCruz™ Staining Systems. The ABC Staining Systems utilizepreformed avidin-biotinylated horseradish peroxidase complex as adetection reagent, whereas the ImmunoCruz™ Staining Systems utilizea streptavidin-horseradish peroxidase complex. The ImmunoCruz™Staining Systems include all secondary reagents in a pre-diluted,ready to use format, and are also available with pre-dilutedprimary antibody included. Complete research protocols are includedwith all Staining Systems; brief protocols are given below.
2. All steps are carried out at roomtemperature in a humidified chamber. Allow all Staining Systemreagents to reach room temperature prior to use. Tissue sectionsshould not be allowed to dry out at any time during the procedure.Use suction to remove reagents after each step, but avoid drying ofspecimens between steps. Use sufficient reagents to cover thespecimens (approximately 100 µl per slide is usually adequate).
ABC Staining Systems
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.
1. Incubate specimens for 1 hour in 1.5%normal blocking serum in PBS (Buffers and General Solutions).Blocking serum (Normal Sera for Immunohistochemistry) ideallyshould be derived from the same species in which the secondaryantibody is raised. Remove blocking serum from slides.
2. Incubate with primary antibody for 30minutes at room temperature or overnight at 4º C. Optimal antibodyconcentration should be determined by titration; recommended rangeis 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.
3. Incubate for 30 minutes withbiotin-conjugated secondary antibody as provided, or atapproximately 1 µg/ml diluted in PBS with 1.5% normal blockingserum. Wash with three changes of PBS for 5 minutes each.
4. Incubate for 30 minutes with avidinbiotin enzyme reagent. Wash with three changes of PBS for 5 minuteseach.
5. Incubate in peroxidase substrate asprovided for 30 seconds–10 minutes, or until desired stainintensity develops. Individual slides should be monitored todetermine the proper development time. Wash sections in deionizedH2O for 5minutes. If desired, counter-stain in Gill´s formulation #2hematoxylin (sc-24973) for 5–10 seconds. Immediately wash withseveral changes of deionized H2O.
6. Dehydrate through alcohols andxylenes as follows: Soak in 95% ethanol twice for 10 seconds each,then 100% ethanol twice for 10 seconds each, then xylenes threetimes for 10 seconds each. Wipe off excess xylene. Immediately add1–2 drops of permanent mounting medium (e.g., Clarion sc-24942),cover with a glass coverslip (sc-24975) and observe by lightmicroscopy.
ImmunoCruz™ Staining Systems
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.
1. Incubate specimens for 20 minutes in 1–3drops of serum block. Aspirate serum from slides.
2. Immediately add 1–3 drops ofpre-diluted primary antibody. If using a Staining System that doesnot include a pre-diluted primary antibody, dilute primary antibodyin serum block to 0.5–5.0 µg/ml as determined by titration.Incubate for 2 hours. Rinse with PBS (Buffers and GeneralSolutions) then wash in PBS twice for 2 minutes each on a stirplate. Aspirate excess liquid from slides.
3. Incubate for 30 minutes in 1–3 dropsof biotinylated secondary antibody. Wash as above.
4. Incubate for 30 minutes in 1–3 dropsof HRP-streptavidin complex. Wash as above.
5. Add 1–3 drops HRP substrate mixture.Develop for 30 seconds–10 minutes, or until desired stain intensitydevelops. Rinse with deionized H2O and transfer to a deionized H2O wash for 2 minutes on a stir plate.
6. Counterstain, dehydrate and mountslides as described under ABC Staining Systems.