Immunofluorescence [IF] Staining (Single:DAPI+1 target)

Immunofluorescence (IF) staining is a widely used technique in biological research and clinical diagnostics. IF utilizes fluorescent-labeled antibodies in order to detect specific target antigens. Followed by imaging, it is a very direct technique as you can actually see something. Although it is a well-established tool, multiple factors have to be considered and various optimization steps have to be taken to ensure successful staining.

Reagent:
In-house

Fluorophore options(choose one):
DAPI (Nuclear)
Alexa Fluor 488
Alexa Fluor 555
Alexa Fluor 594
Alexa Fluor 647
Alexa Fluor 750

Staining Procedure:
1. Deparaffinize and hydrate tissue sections through xylenes or other clearing agents and graded alcohol series.
2. Vector® Antigen Unmasking Solution, Citrate-based, pH 6.0 (H3300) or Tris-based, pH 9.0 (H-3301).
3. Wash the slides with PBS for 5 minutes.
4. Incubate sections for 20 minutes with Normal Goat Serum, 2.5%.
5. Tip off excess serum from sections.
6. Incubate with primary antibody diluted in normal Goat serum overnight at 4°C.
7. Wash slides 2 times with PBS and a 3rd time with PBST, 5 minutes each.
8. Incubate with secondary antibodies diluted in PBS 50min at Room temperature.
9. Wash slides 2 times with PBS and a 3rd time with PBST, 5 minutes each.
10. DAPI stain for 10min
11. Wash the slides with PBS for 3 times, 5 minutes each.
12. Sudan black block background for 5minutes.
13. Running water washes for 20 minutes.
14. Mount