I'm currently staining human bone marrow for:
That's a lot to stain for at one time I know, but so far it's staining well... but only on some samples. Other samples (stained along: same jar, ab mix, at the same time, ect.) do not stain at all. Here's the kicker... even DAPI does not stain on these slides.
Does anyone have any ideas about what could be going on?
I get that samples from a histology lab in a hospital, so unfortunately I have no way of knowing how the slides were treated post biopsy. The samples are fixed in paraffin and cut in the histology lab. They come to me pre melted.
Here's the protocol I'm using:
1. xylenes 2x 5 min.
2. 100% ethanol 2x 3 min.
3. 90% ethanol 2x 3 min.
4. 80% ethanol 2x 3 min.
5. H2O 2x 1 min.
6. Antigen retrieval: 4.5 min. in sodium citrate buffer @ 95C ("happy" medium between optimal time for all antigens)
7. PBS 2x 1 min.
8. Block 2% serum for 1 hour @RT
9. Primary antibody (optimal concentrations in PBS) overnight @4C
10. PBS 2x 5 min.
11. Secondary antibodies (optimal concentrations in PBS) for 1 hour at RT.
12.PBS 2x 1 min.
13. tap H2O 1 min.
14. mount using Vectashield (with DAPI)
Image using confocal.
I spent so much time optimizing antibody concentrations and antigen rescue times. The slides I used to optimize everything were all from the same sample, and it works great. When I moved to patient samples, only 1/3 of the samples stained for anything (normal or disease... doesn't matter).
The key here is that even the DAPI does not work. Meaning it's not an antibody/antigen issue. And, all of the slides have what looks like good nuclei.
Any ideas are appreciated. Iím completely stumped.