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*[[Hosuk:Lab_Notes|LabNote]] ===Human Brain Tissue : Brain Tissue β Fixation condition test=== *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 Jayβs 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==== *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==== *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===== [[File:MIP_HBTissue_Fix37CTest_S1_Fix37C_15min_1stRolony_63x_2k_Pos1_3.png|800px]] =====Compare to previous result : Fix at 25C for 30min :1st Rolonies===== [[File:MIP_HBTissue_2014-12-19_S1_1stRolony_TX100_15m_0.01Pepsin_5min_Croppedfrom20x_4k_Pos3_2.png|400px]] =====Compare to previous result : Fix at 25C for 30min :CA12k_Nov_V4 Padlock Probe===== [[File:MIP_HBTissue_S1_Fix25C_30min_CA12k_Nov_V4_63x_2k_Pos1.png|400px]]
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