I am working on using the dat probe (dopamine transporter), which has been successfully applied to the whole mount in situ hybridization in 5 dpf zebrafish; however, when I applied the same probe on the cryosection, I had a lot of nonspecific background, and was wondering if I could have some suggestions on my protocol?

My current protocol for the adult zebrafish brain is

1. euthanize zebrafish, shave the skull, fixed in 4% PFA, overnight, 4c.

2. remove the whole brain with an additional 4% PFA fixation at RT for 20 mins, and PBS wash 3 times, each 5 min, and transfer to 30% sucrose, overnight, 4c until the brain sinks to the bottom.

3. next day, immerse to OCT: 30% sucrose (2:1) ratio and OCT embedding in liquid nitrogen.

4. Section the zebrafish brain at 10 um, and air-dry the slide at RT for 30 mins before use.

**The cryosection on my IFA protocol worked!

5. PBST (0.1% tween 20) wash, 3 times, each 5 min

6. Proteinase K treatment (10 ug/ml), RT, 10 min

7. Post-fixed with 4% PFA, 15 min

8. PBST wash, 3 times, each 5 min

9. Prehybridize in 5x scc/50% formamide, 65c, 3 hr

10. Hybridization (5X scc, 50% formamide, 100 ug/mg tRNA, 100 ug/ml heparin, 0.1% tween 20), 65c, overnight, probe concentration (0.25 ng-4 ng)

11. wash, 65c, 2x, each 10 min

a. 2x scc/50% formamide

b. 2x scc/25% formamide

c. 2x scc

d. 0.2x scc

12. PBST wash, 2x, each 10 min at RT

13. Blocking, 1hr, RT (2% normal sheep serum, 2mg/ ml BSA, 1X PBST)

14. Anti-DIG AP (1:5000), 4c, overnight

15. PBST wash, 3x, each 5 min

16. Alkaline tris buffer (100 mM Tris HCL, ph9.5, 50 mM Mgcl2, 100 mM NaCl, 0.1% Tween 20), 3x, each 5 min

17. NBT/BCIP coloration

My issues are

1. For both sense and antisense slides, I had a high nonspecific background, which could present 10 min after adding NBT/BCIP (had tried different probe concentrations at 0.25 ng, 0.5 ng, 1 ng, 2 ng, 4 ng/slide)

2. I was thinking about whether it could be because the endogenous AP induced the nonspecific binding, and I tried slides without adding any probes, and no coloration was shown after adding NBT/BCIP; with these results, I was wondering if something was wrong with my probe? for example, RNA degradation, but I have no problem with my whole mount in situ (the RNA stores in the -80c less than 1 month)

3. some protocols use levamisole to inhibit the endogenous AP background, but after trying one slides without any probes, the slide still remained clear, does that mean the nonspecific binding is not related to the endogenous AP?

I was hoping to have more insights and suggestions from your experience. Thank you.