All washes/rinses were performed in cyclindrical glass slide incubators (30mL):
- All SSC washes were pre-heated to appropriate temps before proceeding
- Hybridization solution was discarded and slides rinsed for ~30mins in 2x SSC.
- Cover slips were removed.
- Slides were washed twice in 2x SSC 15mins @ 40C.
- Slides were washed three times in 1x SSC 15mins @ 40C.
- Slides were washed once in 0.5x SSC 15mins @ 40C.
- Tissue was equilibrated in Buffer 1 (100mM Tris-HCl, 10mM NaCl, pH = 7.5) 10mins @ RT.
- Tissues were blocked with Blocking Buffer (Buffer 1 + 2% sheep serum + 0.3% Triton-X 100) for 1hr @ RT (500uL on each slide w/cover slips – sitting flat on bench top).
- Antibody solution (Anti-Digoxigenin-AP Fab fragments [Roche - Exp Nov. 2010]) – Diluted anti-DIG 1:1000 in Blocking Buffer. Anti-DIG is stored @ 4C in FSH 240 on Lisa’s shelf.
- Added 1mL of antibody solution to each slide and incubated without a cover slip for 2hrs @ RT – sitting flat on bench top.
- Rinsed slides with Buffer 1 for 10mins, two times.
- Rinsed slides with Buffer 2 (100mM tris-HCl, 100mM NaCl, 50mM MgCl2; pH = 9.5) for 10mins.
- Incubated slides in substrate solution (30mL Buffer 2 + 135uL NBT [Roch - Exp Feb 2010 - 4-nitro blue tetrazolium] + 75uL BCIP [Roche Exp Nov 2010 - 5-Bromo-4-chloro-3-indolyl phosphate]) O/N @ RT in the dark. Both NBT & BCIP are stored @ -20C in FSH 236 in the “ISH Reagents” box.