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==Imaging Results== *RNAFISH1: Could see faint signal in 488 channel but nothing specific in 594 channel **Fainter signal in 488 compared to [[Matt:LabNotes/2015-6-30 | Third Try w/ cooled CCD camera]] is expected since only 24probes versus 48probes **Failure to see bright distinct spots in 594 channel but instead see bright regions of cells that correspond to dense dark areas in phase channel ***My guess is that dye-probes or maybe free dyes got trapped? *RNAFISH2: Few spots in 488 representing CUX2 transcripts in Alex's neurons **I believe these neurons express less CUX2 than Sebastian's (based on FPKM~2 in Alex's RNA-Seq data and Sebastian claiming CUX2 is "highly expressed" in his neurons) *RNAFISH3: Results similar to RNAFISH1 *RNAFISH4: CUX2 spots are detected but SNAP25 (which should have high expression) is not detected **The Alexa594 channel (SNAP25) shows the same bright regions of cells as the other dishes with 594 channel ***This suggests the bright regions is not due to probe sequence but property of the dye *Conclusion: **Only CUX2-488 probes have worked and there is less CUX2 in Alex's iNGN bipolar neurons than Sebastian's motor neurons **CUX2-594 and SNAP25-594 both exhibited similar peculiarities and suggests the issue lies with Alexa594 dye or Alexa594 dye-coupling (See dye:probe ratio from dye-coupling results) **Need to call LifeTech ARES or Biosearch Stellaris customer support for advice on dye-coupling and bright regions **Next experiment should try switching probe and dye combinations (CUX2-488 and SNAP25-594) ===Images=== ====RNAFISH1==== *CUX2-488 [[File:4_w2Alexa488-Matt.jpg]] *CUX2-594 [[File:4_w1Alexa594-Matt.jpg]] ====RNAFISH2==== *CUX2-488 [[File:RNAFISH2_20150709_4_w2Alexa488-Matt.jpg]] ====RNAFISH3==== *CUX2-488 [[File:RNAFISH3_20150709_8_w2Alexa488-Matt.jpg]] *CUX2-594 [[File:RNAFISH3_20150709_8_w1Alexa594-Matt.jpg]] *Phase [[File:RNAFISH3_20150709_8_w4Phase-WPZ.jpg]] ====RNAFISH4==== *CUX2-488 [[File:RNAFISH4_20150709_9_w2Alexa488-Matt.jpg]] *SNAP25-594 [[File:RNAFISH4_20150709_9_w1Alexa594-Matt.jpg]] *Phase [[File:RNAFISH4_20150709_9_w4Phase-WPZ.jpg]] ====Settings==== *7/10/2015 *Dr. Bern's Zeiss microscope SERF 295 **Light source: X-Cite 120LED **Objective: 100X NA=1.4 oil **Camera: QuantEM 512SC EMCCD Camera **Cubes: Color (Ex filter - Dichroic mirror - Em filter) ***DAPI - (350/50 - 400 - 460/50) ****5% intensity 25msec exposure ***FITC (Alexa488)- (480/30 - 510 - 535/40) ****100% intensity 5sec exposure ***dsRED (Alexa594) - (545/30 - 570 - 628/52) ****20% intensity 150msec exposure ===StarSearch===
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