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Immunofluorescence Protocol: Free Floating Section

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


  • 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


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.


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.


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.

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