Immunofluorescence Protocol: Stick Section

Note: This method is suitable for immunofluorescence on tissue sample.

Reagents of Immunofluorescence Protocol

• PBS: To prepare 1 L 1X PBS: dissolve 8g of NaCl, 0.2g of KCl, 1.44g of Na₂HPO₄, 0.24g of KH₂PO₄ in 800ml distilled H₂O, then adjust pH to 7.4 with HCl and adjust volume to 1L with additional distilled H₂O.
• Permeablization solution: 0.3% Triton X-100 in PBS. To prepare 100 mL: add 300 µl Triton X-100 to 100 mL PBS and mix. (Optional)
• Blocking buffer: To prepare 10 ml, add 0.5 ml normal serum in PBS and mix well. While stirring, add 30 µl Triton™ X-100.
  NOTE: Select serum that are from the same species as the secondary antibody.
• Antibody dilution buffer: To prepare 10 ml, add 30 µl Triton X-100 to 10 ml 1X PBS. Mix well then add 0.1g BSA.
• Antifade Reagent

Steps of Immunofluorescence Protocol

Section Preparation of Immunofluorescence Protocol:

1. The tissue is sliced to ≤10 µm sections and directly mount to adhesive slides.

2. Air dry for 1 hour.

3. Rinse three times in 1X PBS for 5 min each.

4. Drawing a water repellent circle around sections.

5. Sections are incubated in permeablization solution for 3~5 min at room temperature.

NOTE: This step is optimized for intracellular staining.

Blocking of Immunofluorescence Protocol:

6. Shake off the permeablization solution and draw a cycle around sections with PAP pan to keep a liquid pooled in a single droplet.

7. Drop blocking buffer in the cycle and incubate 1 hour at room temperature.

Figure 1. Diagram of water repellent circle

Staining of Immunofluorescence Protocol:

8. Dilute the primary antibody to the recommended concentration on the datasheet in antibody dilution buffer.

9. Shake off the blocking buffer and drop into the cycle with primary antibody solution and incubate overnight at 4°C.

NOTE: If using primary antibodies directly conjugated with fluorochrome, the incubation should be performed in the dark, and then skip to step 12.

10. Rinse three times in 1X PBS for 5 min each.

11. Prepare fluorochrome-conjugated secondary antibody diluted in antibody dilution buffer and incubate for 2~3 hours at room temperature in the dark.

12. Rinse three times in 1X PBS for 5 min each.

13. Coverslip with anti-fade mounting medium.

14. Store at 4°C protecting from light.