Hosuk:LabNotes/2015-2-27
Jump to navigation
Jump to search
Human Brain Tissue : Brain Tissue – Fixation condition test[edit]
- I need to get rolony signal from cytoplasm (outside of nucleus) as much as possible, I still have a question why my samples have much more localized inside nucleus instead of cytoplasm, which seems not correct.
- In Jay’s Science paper (2013), fixing at 37C showed more evenly distributed signals than at 25C. (Somehow they stick to fixing at 25C in Nature protocol)
- So, I try fixing at 37C for 15 min and 30 min, with FISSEQ_RT primer to make 1st rolonies, and see the signals where they are localized.
Procedure[edit]
- Sample : BA8 on Vectabond treated cover glass
- Tissues were hit on 50C hotplate for 3min to remove moisture.
- Fix : 4% Formaldehyde in 1x DEPC-PBS at 37C for 15min
- 0.25% TX-100 for 10min, then 0.01% Pepsin in 0.1N HCl for 5min
- Rest of process is identical (I use FISSEQ_RT primer)
Result[edit]
- Many rolonies were found in cytoplasm, seems well distributed.
- 37C fixation looks effective.
- Of course those signals do not come from targeted padlock probes, so I need to test again with CA12k_Nov2014 padlock probes.
Fix at 37C for 15min :1st Rolonies[edit]
File:MIP HBTissue Fix37CTest S1 Fix37C 15min 1stRolony 63x 2k Pos1 3.png
Compare to previous result : Fix at 25C for 30min :1st Rolonies[edit]
File:MIP HBTissue 2014-12-19 S1 1stRolony TX100 15m 0.01Pepsin 5min Croppedfrom20x 4k Pos3 2.png
Compare to previous result : Fix at 25C for 30min :CA12k_Nov_V4 Padlock Probe[edit]
File:MIP HBTissue S1 Fix25C 30min CA12k Nov V4 63x 2k Pos1.png