Éva MezeyNational Institute of Neurological Disorders and Stroke
National Institutes of Health
http://intramural.nimh.nih.gov/lcmr/snge/Protocols/ychrom.html on fresh frozen sections in the mouse
Twelve ?m thick sections that were previously cut in a cryostat are kept frozen at ?80° C until used.
Remove slides with the sections from freezer and fix in 4% formaldehyde for 10 min at RT.
Wash sections in PBS and in 1xSSC for 10 min each at RT.
Cover sections with 70% formamide/2xSSC and transfer onto a hotplate at 80° C for 10 min to melt the nuclear DNA.
Remove the prehybridization solution (stand up the slides and tap on a filter paper) and cover the sections with the probe solution
For each 25x30mm area: 1 µl of DIG-labelled Y probe added to 2 µl RNA mix, 1 µl SDS, 1 µl NTS and 45 µl of hybridization buffer (see hybridization histochemistry protocol) that is kept at 65° C for 10 min just before use
A 1.5 KB long riboprobe is generated from a plasmid containing a repeat sequence of the mouse Y chromosome (pY3531B) [see C. E. Bishop, P. Boursot, B. Baron, F. Bonhomme, D. Hatat, Nature 315, 70 (1985)]. The riboprobe is produced using T7 polymerase (Ambion Maxiscript Kit) and DIG-UTP (Roche Pharmaceuticals). The DIG labeling is described in the section.RNA (riboprobe) labeling
Put back on the hot plate for 10 more minutes before transfer to an oven where they are hybridized overnight at 55° C
Next morning the sections are washed as described for Dig labelled RNA probes, and transferred into a blocking agent (1xPBS/1%BSA and 0.6% TritonX100) for 15 min at RT.
They are then incubated with an anti-DIG-POD conjugate (Roche) at a dilution of 1:500 in the above blocking agent for 60 min at RT.
Next, a Tyr-FITC conjugate is added according to the manufacturers instructions (PerkinElmer Life Sciences, Inc. - NEN) for 10 min at RT.
After several washes in 0.1M Tris buffer , the sections are viewed under a fluorescent microscope.
Double labeling: For double labeling with an antigenic marker (antibody) and the Y chromosome, the immunostaining is performed first in which the biotinylated tyramide (see immunohistochemistry protocol) is deposited in the tissue. This deposit is insoluble in water and survives the harsh washes and temperatures that are required for the ISHH. After the ISHH is carried out, the deposited biotin can be visualized by adding a Streptavidin-fluorochrome conjugate.
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