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).
Antibody solution – Diluted alkaline phosphatase-labelled sheep anti-DIG 1:1000 in Blocking Buffer.
Added 1mL of antibody solution to each slide and incubated without a cover slip for 2hrs @ RT.
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 (10mL Buffer 2 + 45uL NBT [nitroblue tetrazolium] + 25uL BCIP) O/N @ RT.