IF protocol-Free Floating Section

1. IF 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 2 3 temperature. The tissue is sliced to 20~30 µm free floating sections. Rinse three times in 1X PBS for 5 min each. Sections are incubated in permeablization solution for 3~5 min at room NOTE: This step is optimized for intracellular staining. https://www.creative-diagnostics.com/immunofluorescence-protocol-free-floating-s ection.htm