Immunofluorescence Protocol for Cell-based Imaging (Immunocytochemistry)

IMPORTANT: Please refer to the Product Usage Information section on the product webpage or datasheet to determine if your product is validated and approved for use on cultured cell lines (IF-IC).

A. Solutions and Reagents

Achieve higher quality immunofluorescent images using the efficient and cost-effective, pre-made reagents in our Immunofluorescence Application Solutions Kit #12727.

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

1. 1X Phosphate Buffered Saline (PBS): To prepare 1L of 1X PBS, add 100 mL 10X Wash Buffer, Phosphate Buffered Saline #12528 to 900 mL dH₂O, mix. Adjust pH to 8.0.
2. Product-specific Reagents
   1. 4% Formaldehyde, Methanol-Free #47746: Use fresh.
   2. 100% Methanol #13604: Chill before use.
3. Blocking Buffer: Purchase ready-to-use Immunofluorescence Blocking Buffer #12411, or prepare a 1X PBS / 5% normal serum / 0.3% Triton X-100 buffer by adding 0.5 mL normal serum from the same species as the secondary antibody (e.g., Normal Goat Serum #5425) and 30 µL Triton X-100 to 9.5 mL of 1X PBS. Store at 4°C.
4. Antibody Dilution Buffer: Purchase ready-to-use Immunofluorescence Antibody Dilution Buffer #12378, or prepare a 1X PBS / 1% BSA / 0.3% Triton X-100 buffer by adding 0.1 g BSA #9998 and 30 µL Triton X-100 to 10 mL of 1X PBS. Store at 4°C.
5. Fluorochrome-conjugated Secondary Antibody: Use a secondary antibody that is reactive to your primary antibody host species (e.g., rabbit). Click here for a list of secondary antibodies approved for immunofluorescence.
6. Mounting Medium: Use Prolong Gold AntiFade Reagent #9071 or Prolong Gold AntiFade Reagent with DAPI #8961.

B. Sample Preparation

IMPORTANT: CST^® antibodies require specific sample preparation steps for optimal results. Please refer to the protocol on the product webpage for detailed recommendations.

• Fixation
  • Formaldehyde Fixation: Fix cells with 4% formaldehyde for 15 min at room temperature.
  • Methanol Fixation: Gently and drop-wise, cover cells with ice-cold 100% methanol and then incubate for 15 min at –20°C or on ice. Do not allow samples to dry.
• Wash: Aspirate fixative and then wash three times in 1X PBS for 5 min each.
• Permeabilization (after Formaldehyde Fixation)
  • Triton X-100 Permeabilization: Proceed to Section C, Step 1.
  • Methanol Permeabilization: Cover cells with ice-cold 100% methanol and then incubate for 10 min at –20°C or on ice. Aspirate methanol and then wash three times in 1X PBS for 5 min each. Do not allow samples to dry.

C. Immunostaining

1. Block specimen with Blocking Buffer for 60 min.
2. While blocking, prepare each primary antibody by diluting it in Antibody Dilution Buffer as indicated on the product webpage or datasheet.
3. Aspirate blocking solution and then apply diluted primary antibody.
4. Incubate overnight at 4°C.
5. Wash three times with 1X PBS for 5 min each.

NOTE: If using a fluorochrome-conjugated primary antibody, then skip to Step 8.

6. Incubate specimen with fluorochrome-conjugated secondary antibody diluted in Antibody Dilution Buffer for 1–2 hr protected from light.
7. Wash three times in 1X PBS for 5 min each.
8. Counterstain as appropriate and then mount samples for imaging.
9. For long-term storage, store samples at 4°C protected from light.

Contact us with any questions.

For Research Use Only. Not for Use in Diagnostic Procedures.