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Antibody staining on Sections

Jared Talbot, Ruth A BreMiller and Yi-Lin Yan

Fixation and section preparation:

1. Raise fish to desired stage.

2. Fix overnight in 4% PFA in 1X PBS.

3. Wash out of fix over ~30 minutes total

Wash 3X in PBST

Wash 3X in PBS

Note: fish can be dehydrated in PBS:methanol series (3:1 of PBS : methanol; 1:1 of PBS : methanol; 1:3 of PBS : methanol; 100% methanol) and stored in 100% methanol at -20˚C for a period of time and rehydrated in the PBS:methanol series (1:3 of PBS:methanol; 1:1 of PBS:methanol;  3:1 of PBS: methanol) and then washed with PBS for a few times before imbedding for sectioning.

4. Imbed fish in agar/agarose.

5. Leave the fish agar/agarose block in 30% sucrose overnight at 4˚C.

6. Trim agar/agarose block, freeze slowly in liquid nitrogen.

7. Section tissues; 4 heads/slide, 16µm sections, on Starfrost slides.

8. After sectioning, leave slides out at room temperature overnight (O/N).

9. Under dissecting scope, select slides with optimal sections. Set aside slides with optimal sectioning.

10. Seal boxes with tape.

11. Store at -20˚C.

Antibody staining:         

Day 1:

1. Remove slides from freezer and thaw to room temperature.

2. Ring all slides carefully with Pap Pen.

3. Wash 3X in PBST

4. Prepare horizontal holder:

2 blot papers, soaked with PBST, and two matched risers.

5. Blot off slides. Immediately place in horizontal holder.

6. Add 500 µl block solution to slides inside PAP ring.

7. Place at 4˚C for at least four hours.

8. Prepare primary antibodies in block solution.

9. Add 100 µl primary antibody in block solution to the slides. Cover slides with parafilm, and ensure that there are no bubbles.

10. Place at 4˚C overnight.

Day 2:

11. Remove Parafilm.

12. Wash 6X in PBST over at least one hour total.

13. Add 500µl block solution for at least 1 hour.

14. Prepare secondary antibodies in block solution at desired dilution.

15. Remove block solution.

16. Add 200µl secondary antibody in block solution to each slide. Cover with parafilm.

17. Leave at 4˚C O/N (or at room temperature for a few hours).

 Day 3:

18. Wash 4X in PBST for 10 min each.

19. Add nuclear stain, TO-PRO®-3 Iodide (Life Technology, Cat: T3905), 1:10,000 dilution in PBS.

20. Wash 6x in PBST 5 min each.

21. Add 60µl of 50% glycerol or 2 drops of SlowFade Gold Antifade  (Life Technology, Cat#: S36973 ), or ProLong Gold Antifade (Life Technology , Cat#: P36934) , then add coverslide.

22. Seal slides with fingernail polish.

23. Store at 4˚C before imaging.

Agar/agarose:

0.9%  agar

1.0%  low melt agarose

5%  sucrose

PBST:

PBS plus 0.1% Tween 20

Block solution:

10X PBS                               2.5 ml

Normal goat serum               1 ml

Normal sheep serum            500 µl

20% tween,                           250 µl

sterile H2O                              20.8 ml