Immunofluorescence [IF] Staining (2-plex:DAPI+2 targets)
$50/slide (If antibody is provided by iHisto, an additional cost of $10/antibody)
Click the image to view whole slide scanning
Figure: Insulin antibody labeled Alexa 594 and Glucagon antibody labeled Alexa 488
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.
Fluorophore options(choose two):
Alexa Fluor 488
Alexa Fluor 555
Alexa Fluor 594
Alexa Fluor 647
Alexa Fluor 750
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.