PROTOCOLS

7: Immunofluorescence Cell 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.
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.
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).
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.
D. Immunofluorescence Staining
Use suction to remove reagents after each step, but avoid drying of specimens between steps. Use sufficient reagent to cover the specimen (approximately 100–500 µl per slide is adequate).
Incubate specimens with 10% normal blocking serum (Normal Sera for Immunohistochemistry) in PBS (Buffers and General Solutions) for 20 minutes to suppress non-specific binding of IgG. Blocking serum ideally should be derived from the same species in which the secondary antibody is raised. Wash with PBS.
Incubate with primary antibody for 60 minutes. Optimal antibody concentration should be determined by titration; recommended range is 0.5–5.0 µg/ml in PBS with 1.5% normal blocking serum. Wash with three changes of PBS for 5 minutes each.
Incubate for 45 minutes with either biotin-conjugated or fluorochrome-conjugated secondary antibody (Secondary Antibodies for Immunohistochemistry) diluted to 1–5 µg/ml in PBS with 1.5%–3% normal blocking serum. Optimal antibody concentration should be determined by titration. Wash with three changes of PBS. If fluorochrome-conjugated secondary antibody is used, incubate in a dark chamber and omit the next step.
Incubate with streptavidin-fluorescein for 15 minutes in a dark chamber. Optimal streptavidin conjugate concentration for a given application should be determined by titration; recommended range is 10–20 µg/ml in PBS. Wash extensively with PBS.
Mount coverslip with aqueous mounting medium or 90% glycerol in PBS.
NOTE: For a listing of mounting media for Immunohistochemistry including Organo/Limonene and UltraCruz™ Mounting, see Mounting Media for Immunohistochemistry.
Examine using a fluorescence microscope with appropriate filters. Store slides in a dark location at room temperature (UltraCruz™ Mounting Medium: sc-24941) or at 4º C (glycerol/PBS mount).
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Immunofluorescence Cell Staining
Isotype-Specific Secondary Antibodies for Immunohistochemistry

Control IgGs and IgG Conjugates

Conventional Secondary Antibodies for Immunohistochemistry

F(ab')2 Secondary Antibodies for Immunohistochemistry

Mounting Media for Immunohistochemistry

Normal Sera for Immunohistochemical Blocking Applications

Isotype-Specific Secondary Antibodies

F(ab')2 Secondary Antibodies

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