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