Insitu

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...