Immunofluorescence Protocol

Product Manager: Harrison Michael​

Important Notes:  This protocol is applicable to both unlabeled and fluorescently labeled antibodies. Please refer to the product usage information section on the product webpage or product data sheet to confirm whether the product has been validated and approved for use in cultured cell lines (IF-IC) or frozen tissue sections (IF-F).

Note: Some Aladdin antibodies perform optimally with specific alternative protocols. Please refer to the product webpage for recommended protocols for specific products.​​

I. Immunofluorescence Protocol with Formaldehyde Fixation

A. Solutions and Reagents

Note: Prepare solutions using reverse osmosis deionized water (RODI) or water of equivalent quality.

· 1 × Phosphate Buffered Saline (PBS): To prepare 1 L of 1× PBS: Add 100 mL of 10× washing buffer (Phosphate Buffered Saline, P492453) to 900 mL of dH₂O, mix well. Adjust pH to 8.0.

· 4% Formaldehyde, methanol-free: Use a fresh solution.

· Blocking Buffer: Prepare 1× PBS / 5% normal serum / 0.3% Triton X-100 buffer by adding 0.5 mL of normal serum from the same species as the secondary antibody (e.g., Normal Goat Serum, G752123) and 30 µL of Triton X-100 (T109027) to 9.5 mL of 1× PBS. Store at 4°C.

· Antibody Dilution Buffer: Prepare 1× PBS / 1% BSA / 0.3% Triton X-100 buffer by adding 0.1 g of BSA (B265993) and 30 µL of Triton X-100 to 10 mL of 1× PBS. Store at 4°C.

· Fluorescein-conjugated secondary antibody: Use a secondary antibody reactive with the host species of your primary antibody (e.g., rabbit). Click here for a list of secondary antibodies approved for immunofluorescence.

B. Fixation

Note: All subsequent incubations should be performed at room temperature (20-25°C) unless otherwise specified.

1. For immunostaining of fixed frozen tissues (IF-F), proceed to Section C.

2. For cultured cell lines (IF-IC) or unfixed frozen tissue sections (IF-F), fix immediately as follows:

a. Cover the sample with 4% formaldehyde to a depth of 2–3 mm.

b. Allow the specimen to fix at room temperature for 15 minutes.

c. Rinse three times with PBS, 5 minutes each.

d. Proceed to immunostaining (Section C).

C. Immunostaining

1. Block the specimen in blocking buffer for 60 minutes.

2. While blocking, prepare the primary antibody in antibody dilution buffer (see product website for recommended dilution range).

3. Aspirate the blocking solution and add the diluted primary antibody.

4. Incubate overnight at 4°C.

5. Rinse three times with 1× PBS, 5 minutes each.

Note: If using a fluorescently labeled primary antibody, skip to Step 8 of Section C.

6. Dilute the fluorescein-conjugated secondary antibody in antibody dilution buffer, then incubate the specimen in the dark for 1–2 hours.

7. Rinse three times with 1× PBS, 5 minutes each, while protecting from light.

8. Perform appropriate counterstaining.

Note: When adding fluorescent cell dyes (including DNA dyes, etc.) to your experiment, refer to the dye product page for recommended protocols. View our list of cell dyes validated for immunofluorescence assays.​

9. Mount the sample for imaging.

10. For long-term storage, store the sample at 4°C in the dark.

II. Immunofluorescence Protocol for Cell-Based Assays Requiring Methanol Fixation

A. Solutions and Reagents

Note: Prepare solutions using reverse osmosis deionized water (RODI) or water of equivalent quality.

1. 1× Phosphate Buffered Saline (PBS): To prepare 1 L of 1× PBS: Add 100 mL of 10× washing buffer (Phosphate Buffered Saline,P492453) to 900 mL of dH₂O, mix well. Adjust pH to 8.0.

2. Methanol (M116124): 100%, use ice-cold solution.

3. Blocking Buffer: Purchase ready-to-use Immunofluorescence Blocking Buffer (B751639), or prepare 1× PBS / 5% normal serum / 0.3% Triton X-100 buffer by adding 0.5 mL of normal serum from the same species as the secondary antibody (e.g., Normal Goat Serum, G752123) and 30 µL of Triton X-100 (T109027) to 9.5 mL of 1× PBS. Store at 4°C.

4. Antibody Dilution Buffer: Prepare 1× PBS / 1% BSA / 0.3% Triton X-100 buffer by adding 0.1 g of BSA (B265993) and 30 µL of Triton X-100 to 10 mL of 1× PBS. Store at 4°C.

5. Fluorescein-conjugated secondary antibody: Use a secondary antibody reactive with the host species of your primary antibody (e.g., rabbit). Click here for a list of secondary antibodies approved for immunofluorescence.​

B. Fixation

Note: All subsequent incubations should be performed at room temperature (20-25°C) unless otherwise specified.

1.Aspirate the medium, then cover the cells with ice-cold 100% methanol to a depth of 2–3 mm.

2.Allow the cells to fix on ice or at 4°C for 15 minutes.

3.Rinse three times with 1× PBS, 5 minutes each.

4.Proceed to immunostaining (Section C).

C. Immunostaining

1. Block the specimen in blocking buffer for 60 minutes.

2. While blocking, prepare the primary antibody in antibody dilution buffer (see product website for recommended dilution range).

3. Aspirate the blocking solution and add the diluted primary antibody.

4. Incubate overnight at 4°C.

5. Rinse three times with 1× PBS, 5 minutes each.

Note: If using a fluorescently labeled primary antibody, skip to Step 8 of Section C.

6. Dilute the fluorescein-conjugated secondary antibody in antibody dilution buffer, then incubate the specimen in the dark for 1–2 hours.

7. Rinse three times with 1× PBS, 5 minutes each, while protecting from light.

8. Perform appropriate counterstaining.

Note: When adding fluorescent cell dyes (including DNA dyes, etc.) to your experiment, refer to the dye product page for recommended protocols. View our list of cell dyes validated for immunofluorescence assays.​

9. Mount the sample for imaging.

10. For long-term storage, store the sample at 4°C in the dark.

III. Immunofluorescence Protocol with Methanol Permeabilization

A. Solutions and Reagents

Note: Prepare solutions using reverse osmosis deionized water (RODI) or water of equivalent quality.

1. 1× Phosphate Buffered Saline (PBS): To prepare 1 L of 1× PBS: Add 100 mL of 10× washing buffer (Phosphate Buffered Saline, P492453) to 900 mL of dH₂O, mix well. Adjust pH to 8.0.

2. 4% Formaldehyde, methanol-free: Use a fresh solution.

3. Methanol (M116124): 100%, use ice-cold solution.

4. Blocking Buffer: Purchase ready-to-use Immunofluorescence Blocking Buffer (B751639), or prepare 1× PBS / 5% normal serum / 0.3% Triton X-100 buffer by adding 0.5 mL of normal serum from the same species as the secondary antibody (e.g., Normal Goat Serum, G752123) and 30 µL of Triton X-100 (T109027) to 9.5 mL of 1× PBS. Store at 4°C.

5. Antibody Dilution Buffer: Prepare 1× PBS / 1% BSA / 0.3% Triton X-100 buffer by adding 0.1 g of BSA (B265993) and 30 µL of Triton X-100 to 10 mL of 1× PBS. Store at 4°C.

6. Fluorescein-conjugated secondary antibody: Use a secondary antibody reactive with the host species of your primary antibody (e.g., rabbit). Click here for a list of secondary antibodies approved for immunofluorescence.​

B. Fixation

Note: All subsequent incubations should be performed at room temperature (20-25°C) unless otherwise specified.

1. For immunostaining of fixed frozen tissues (IF-F), proceed to Section C.

2. For cultured cell lines (IF-IC) or unfixed frozen tissue sections (IF-F), fix immediately as follows:

a. Cover the sample with 4% formaldehyde to a depth of 2–3 mm.

b. Allow the specimen to fix at room temperature for 15 minutes.

c. Rinse three times with PBS, 5 minutes each.

d. Proceed to immunostaining (Section C).

C. Immunostaining

Note: All subsequent incubations should be performed at room temperature unless otherwise specified. Incubate in a humid, light-tight container or covered dish/plate to prevent drying and fluorophore quenching.

1. Methanol permeabilization step: Cover the sample with ice-cold 100% methanol to a depth of 2–3 mm, then incubate on ice or at 4°C for 10 minutes.

2. Rinse three times with 1× PBS.

3. Block the specimen in blocking buffer for 60 minutes.

4. While blocking, prepare the primary antibody in antibody dilution buffer (see product website for recommended dilution range).

5. Aspirate the blocking solution and add the diluted primary antibody.

6. Incubate overnight at 4°C.

7. Rinse three times with 1× PBS, 5 minutes each.

Note: If using a fluorescently labeled primary antibody, skip to Step 10 of Section C.

8. Dilute the fluorescein-conjugated secondary antibody in antibody dilution buffer, then incubate the specimen in the dark for 1–2 hours.

9. Rinse three times with 1× PBS, 5 minutes each, while protecting from light.

10. Perform appropriate counterstaining.

Note: When adding fluorescent cell dyes (including DNA dyes, etc.) to your experiment, refer to the dye product page for recommended protocols. View our list of cell dyes validated for immunofluorescence assays.​

11. Mount the sample for imaging.

For long-term storage, store the sample at 4°C in the dark.

Aladdin：https://www.aladdinsci.com/