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All steps are performed in accordance with BioLegend's Immunocytochemistry Staining Protocol.
Step-by-step immunocytochemistry protocol by BioLegend covering cell preparation, fixation, staining, and mounting for microscopy imaging.
Key Takeaways
- Proper coverslip preparation and cell plating are critical for successful immunocytochemistry.
- Fixation and permeabilization steps must be optimized based on intracellular or surface staining needs.
- Centrifugation of antibody solutions prevents aggregates and improves staining quality.
- Blocking reduces nonspecific antibody binding, enhancing signal specificity.
- Careful mounting and sealing preserve fluorescence for microscopy imaging.
Summary
- Sterilize coverslips with 70% ethanol and optionally coat with Poly-D-Lysine for cell attachment.
- Detach cells, centrifuge, resuspend, and plate on coverslips, growing overnight to 70-80% confluency.
- Fix cells with 1% paraformaldehyde, then permeabilize for intracellular staining using 0.5% PBST or Triton X-100.
- Block nonspecific binding with 5% FBS in PBS before antibody incubation.
- Centrifuge diluted primary antibody to remove aggregates and incubate 2-3 hours at room temperature or overnight at 4°C.
- Perform washes in PBS between staining steps; use different protocols for surface vs intracellular staining.
- If primary antibody is conjugated, mount coverslips with anti-fade medium and seal with nail polish.
- For secondary antibody staining, incubate with fluorochrome-conjugated secondary antibody after primary antibody wash.
- Optional F-actin staining with Flash Phalloidin™ for 20 minutes in the dark.
- Final mounting of coverslips on slides is done carefully to avoid bubbles before imaging under a microscope.
Full Transcript — Download SRT & Markdown
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Prepare cover slips by sterilizing with 70% ethanol for a minimum of 20 minutes at room temperature.
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Rinse cover slips 3 times in PBS.
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Optional: For loosely attaching cells, add 1 mL of 0.1 mg/mL Poly-D-Lysine.
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Incubate for 20 minutes at room temperature (under UV light, if possible).
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Wash three times with PBS and dry.
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Rinse cells with PBS.
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Detach cells according to your cell type and centrifuge at 400 x g for 5 minutes.
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Discard the supernatant.
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Resuspend the cells in media to the appropriate concentration for your cell type.
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Gently plate 1 mL of cells in each well containing a coverslip.
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Grow cultured cells overnight in a 12-well plate at 37°C.
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At time of fixation, cells should be ~70-80% confluent in single layer.
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Rinse cells in PBS.
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Fix cells with freshly made 1% paraformaldehyde in PBS for 10 minutes at room temperature.
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Rinse 3 times in PBS.
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For intracellular staining: Permeabilize in 0.5% PBST (or Triton X-100 in PBS) at room temperature.
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Incubate for 10 minutes.
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Rinse 3 times in PBS.
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Block in 1 mL of 5% FBS in PBS at room temperature for 30 minutes.
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Dilute primary antibody in 5% FBS in PBS.
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If the primary antibody is conjugated, centrifuge the diluted antibody at max speed for 10 minutes to avoid any aggregates.
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Add 500 µl per well, incubate for 2-3 hours at room temperature, or overnight at 4°C (in the dark if conjugated).
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For surface staining, Rinse 3 times in PBS. For intracellular staining: Rinse in PBS for 10 minutes at 4°C followed by two quick washes in PBS.
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If primary antibody is directly conjugated: Add anti-fade mounting medium to slide.
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Take out the cover glass from well.
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Carefully mount the cover slip on the slide, cell-side down, avoiding any bubbles.
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Seal slides with nail polish.
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If using fluorochrome-conjugated secondary antibody: Prepare the secondary antibody in 5% FBS in PBS.
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Centrifuge the diluted antibody at max speed for 10 minutes to avoid any aggregates.
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Add 500 µl per well, and incubate for 1 hour at room temperature, in the dark.
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Rinse in PBS for 10 minutes at 4°C followed by two quick washes in PBS.
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Optional: To stain F-actin, prepare a working solution of Flash Phalloidin™ by diluting it 1:20 - 1:100 in PBS.
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Add 500 µL and stain for 20 minutes at room temperature in the dark.
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Add anti-fade mounting medium to slide.
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Take out the cover slip from well.
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Carefully mount the cover glass on the slide, cell-side down, avoiding any bubbles.
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Seal slides with nail polish and image on a microscope.
Topics:immunocytochemistryBioLegendcell stainingantibody stainingfluorescence microscopycell fixationpermeabilizationF-actin stainingPoly-D-Lysineimmunostaining protocol
