Immunofluorescence Protocol: Free Floating Section

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

Reagents:

• 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:

Section preparation:

1   The tissue is sliced to 20~30 µm free floating sections.
2   Rinse three times in 1X PBS for 5 min each.
3   Sections are incubated in permeablization solution for 3~5 min at room temperature.
      NOTE: This step is optimized for intracellular staining.

Blocking:

4   Add 200µl blocking buffer to 24 well (or an eppendorf tube) of the chamber sections and incubate 60 min at room temperature.
      NOTE: The volume of buffer could be varying according to the size and quantity of sections.

Staining:

5   Dilute the primary antibody to the recommended concentration on the datasheet in antibody dilution buffer.
6   Add 200µl per well to the chamber slides and incubate two to three hours on a shaker at room temperature or 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 9.
7   Rinse three times in 1X PBS for 5 min each.
8   Prepare fluorochrome-conjugated secondary antibody diluted in antibody dilution buffer and incubate for 2~3 hours at room temperature in the dark.
9   Rinse three times in 1X PBS for 5 min each.
10  Mount the sections on to slides and coverslip with anti-fade mounting medium.
11 Store at 4°C protecting from light.

Learn the principle of immunofluorescence.

Learn immunofluorescence staining on stick section and cultured cells.

If you have any question, please contact us at: info@creative-diagnostics.com.

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