Submitted by: Submitted by ktmess
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Category: Science and Technology
Date Submitted: 10/22/2013 08:08 AM
ISH v.2 In Situ #6 Started On:
Goal:
Colormetric in situ aimed to test different amounts of time 174-9 MOE are post fixed in 4% PFA.
Probe:
T-3 antisense probe: 340 nanograms/micro liter
Tissue:
14 micron 174-9 MOE sections:
• 1 Slide where the tissue has been post fixed at 4 degrees for 1 hour
• 1 Slide where the tissue has been post fixed at 4 degrees for 3 hours
• 1 Slide where the tissue has been post fixed at 4 degrees overnight
All slides were in EDTA overnight at 4 degrees
Day 1
Dry slides at RT, label.
Fix in 4% PFA for 10 minutes.
Wash in DEPC-PBS for 3 X 2 min.
For adult MOE (14 um sections), treat with PK for 10-15 min. at RT
PK solution: 50 mM Tris, pH 7.5, 5 mM EDTA, 10 (g/ml PK
Wash in DEPC-PBS for 5 minutes.
Fix in PFA for 15 minutes.
Rinse in DEPC-dH2O 2 X 5 min.
Add TEA/acetic anhydride for 10 minutes.
TEA/acetic anhydride solution: 1X TEA, 0.25% acetic anhydride
Wash in DEPC-PBS for 3 X 2 min.
Prehyb at RT for 1-2 hours.
Prepare incubator at 64-68°C and heat block at 80°C.
Prepare probe solution (100 (l/slide). Mix well.
Probe solution: Probes in hyb buffer (Ma Lab ISH protocol), 200 ul/slide (probe conc. = 1ng/ul soln to be added to slide)
Heat probe solution at 80°C for 5 min.
Cool on ice for 5 min.
Prepare hyb chamber. Place a paper towel, rolled up and soaked in prehyb solution, in the bottom of a slide box.
Apply 60 (l of probe solution (make sure to mix again) to each slide. Place a 24 x 50 mm coverslip on top. Slide into a slot in the hyb chamber. Place dummy slides with 70 (l of prehyb solution in the slots above and below the test slides.
Incubate at 64-68°C O/N.
Pre-heat 100 ml 2X SSC and 0.2X SSC at 64-68°C O/N.
Time Pre-hybe started:__________ Time Pre-hybe ended:___________
Time Probe incubation started:_________ Time Probe incubation ended:__________
Day 2
Place slides in a mailer with preheated 2X SSC. Incubate at 64-68°C for 5...