>‘O�ˆ—�

section
�«
4�“PFA/PBS  Fix at RT�@15'
�«
wash in PBS 5'x2
�«
ProK in 10mM Tris-Cl(pH8.0)  37�Ž 10'   
(1M Tris 1.5mL + 500mM EDTA 0.3mL + 15mg/mL ProK 75ul)
�«
4% PFA/PBS  Fix at RT�@10'
�«
wash in PBS�@5'x2
�«
0.1M Triethanolamine/HCl  1'   
(5.3mL Triethanolamine + 0.9mL HCl in 400mL H2O)
�«
+1mL acetic anhydride, gently stir, 10' at RT
�«
wash in PBS
�«
air dry

*hybridization B(20x):

�@100% formamide  2mL
�@1M Tris-Cl(7.6)   40ul
�@10mg/mL tRNA   80ul
�@50% dextran   0.8mL
�@50x denhalt's   80ul
�@5M NaCl   480ul
�@10% SDS   100ul
�@0.5M EDTA   8ul
�@dH2O   412ul

1ul flu probe + 1ul DIG probe in 200ul Buf

>Hybridization
51�Ž, 16h in moisture chamber

>wash

5xSSC at 55�Ž in a beaker
�«
50% formamide / 2xSSC, 65�Ž�@15'
�«
2xSSC  65�Ž�@15'
�«
0.2xSSC  65�Ž�@15' x2
�«
PBS 
�«
blocking B   (1% blocking reagent/PBS)   60'
�«
anti-DIG-HRP (Roche) (1/50)
�«
bio-TSA (1/100) + 1/100 0.15% H2O2 in PBS/0.01M Imidazole 20'
�«
HRPŽ¸Šˆ 3% H2O2 in PBS >2h
�«
Rb anti-flu-HRP (PerkinElmer) (1/100) +StAv488 60'
�«
Cy3-TSA (1/100) + 1/100 0.15% H2O2 in PBS/0.01M Imidazole 15'
�«
wash