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