Unless noted otherwise, all incubations take place at room temperature
Heat tissues to 60°C for 10 mins Deparaffinize and rehydrate tissue sections through series of xylenes and ethanol incubations
a. Xylenes or alternative (Citrisolve, Histoclear) incubate 2 times, 5 min each incubation (2 x 5 min)
b. 100% ethanol, 5 min
c. 95% ethanol, 5 min
d. 70% ethanol, 5 min
Rinse the slides once in dH20 Wash with PBS for 5 mins Antigen retrieval: Preheat BD retrievagen A solution (BD Pharmigen cat# 550524) in coplin jar in steamer.
Generate Y chromosome template DNA If you are starting with an aliquot of 2-4 µl template DNA, amplify the DNA several (2-3) times by DOP-PCR to generate Y chromosome template. This will be your Y chromosome template stock. Then each time you make probe use DNA from this stock for another batch (8 reaction) DOP-PCR and subsequent labeling. Do not repeatedly amplify DOP-PCR as this can bias the oligos generated by selecting for a subpopulation of oligos that amplify particularly well.
Protocol for preparation of slides with bone marrow or peripheral blood for fluorescence in situ hybridization
Unless noted otherwise, all incubations take place at room temperature
Equilibrate frozen sections to RT Wash in 2xSSC, incubate two times, 3 min each incubation (2x3min) Incubate in 0.2 M HCL for 10 min at room temperature Wash in 2xSSC 2x3min Incubate in 1M NaSCN for 20 min at 75-80°C Wash in 2xSSC 2x3min Water rinse Ethanol dehydrate (70%, 95%, 100%), 1 min each Air-dry slides Hybridization: pre-heat probe to 75°C, vortex to mix