General Immunostaining protocol

 

Wash è Block è 1° antibody è Wash è 2° antibody è Wash è Post-fix è Wash è Mount è Seal

 

 

* Take care not to let tissue sections dry out


1. Fill a foil-wrapped Coplin jar with cold 1X PBS. Transfer slides immediately from -80°C into jar.

2. Wash with cold 1X PBS 3x5 minutes with shaking.

3. Dry backs of slides and around sections, taking care not to touch sections themselves. Draw a wax circle around each section with ImmEdge Hydrophobic Barrier Pen (Vector Laboratories, H-4000). Label slides according to experiment date for easy reference.

4. From this point on, all steps should be done in the dark.

5. Place slides in a blackout humidified chamber and block each section with 80 μL 10% PBS Blocking Solution, making sure the section is covered entirely.

a. PBS Blocking Solution = 90% 1X PBS + 10% Serum from host of Secondary or Conjugated Primary Antibody.

• Donkey Serum (Jackson ImmunoResearch, 017-000-121)

• Mouse Serum (Jackson ImmunoResearch, 015-000-120)

6. Incubate for 30 minutes at room temperature.

7. Tap off solution, place slides in a blackout humidified chamber, and add 80 μL primary antibody diluted in 10% PBS Blocking Solution, making sure the section is covered entirely. 

• Add 10% PBS Blocking Solution only to Negative Control Section.

8. Incubate at 4°C overnight.

9. Tap off solution, and wash in a foil-wrapped Coplin jar with cold 1X PBS 3x5 minutes with shaking.

10. Place slides in a blackout humidified chamber and add 80 μL secondary antibody diluted in 10% PBS Blocking Solution.

• Add 1X PBS to section with Conjugated Primary Antibody.

11. Incubate for 1 hour at room temperature.

12. Tap off solution, and wash in a foil-wrapped Coplin jar with cold 1X PBS 3x5 minutes with shaking.

13. If double staining, repeat Steps 4-12.

14. Post-fix sections with fresh/frozen 1% PFA for 2 minutes at room temperature.

15. Wash in a foil-wrapped Coplin jar with cold 1X PBS 3x5 minutes with shaking.

16. Mount with 5 μL ProLong Gold with DAPI (Life Technologies, P-36931) by putting three 5 μL droplets on the slide, one near/on each.

17. Place slide facedown on a coverglass (Fisher Scientific, 125485E), avoid bubble and excess liquid accumulation and let rest on a level surface overnight at 4°C.

18. Seal slide/coverglass with nail polish (Fisher Scientific, S30697).

19. Store long-term at 4°C in slide storage box.