I am a radiation oncologist from Turkey and I am studying neurogenesis on hippocampus of rats. Currently my research partner in histology and I are having difficulties with staining sample tissues using CD68 (Santa Cruz), doublecortin (Santa Cruz) and Ki67 (Abcam ab16667) markers
So far we have taken the steps below but unfortunately that didn't yield selective staining. Could you spot anything we possibly may have done wrong?
We also tried skipping step #8. The antibody was diluted 1/100 and we waited overnight at +4°C. Most parts of the tissue were stained but there was no specific staining.
To see if the antibody is working, positive control stain was made in the intestinal sample. The antibody was diluted 1/100 and stood overnight at +4°C. It stained.
After sacrification, we performed intracardiac perfusion fixation, and the tissues were embedded in paraffin within 2 weeks. We have not encountered any problems with cutting tissues with the microtome.
1. The brains of rats were embedded in paraffin after perfusion fixation procedure
2. Sections with 4μ thickness were taken
3. Deparaphinization for 1 night at incubator (at 60ᵒC)
4. Xylene for 30 minutes
5. Dehydration in graded series of alcohols (100%, 90%, 75%, dH2O)
6. Antigen retrieval for 30 min at 37ᵒC trypsin or 0.01M citrate buffer 3x1min, 3x5min (all possibilities have been tried)
7. PBS for 3x5min
8. 3% H2O2 in Methanol x5 min, 1% H2O2 in Methanol x15 min, 3% H2O2 in PBS x5 min (all possibilities have been tried)
9. Blocking solution x 5 min
10. Stained with: DCX (sc390645) diluted 1/100 and 1/50 with PBS, Ki67 (ab16667) diluted 1/100 and 1/50 with PBS, CD68 (sc7084) diluted 1/100 and 1/50 with PBS, 2 hours at room temperature, 1 hour at 37ᵒC or overnight at +4ᵒC (all possibilities have been tried)
11. PBS for 3x5 min
12. Seconder antibody for 10 min
13. PBS for 3x5 min
14.Enzyme-labeled avidin-biotin complex for 10 min
15. PBS for 3x5 min
16. DAB x 1 min, 2 min
17. Washed with dH2O
18. Hematoxylin for 1 min or no hematoxylin staining (all possibilities have been tried)
19. dH2O, 80%,90%, 100% alcohols and xylene
20. Closed with Entellan
We would be grateful if someone can point out where we possibly made a mistake? Thank you in advance.