Modified Fiber FISH detection

This protocol uses a two layer flourescent + anti-body stacking to maximize DNA fiber detection. After step seven be sure to do washes in the dark, and use a 22 X 60 coverslip for the incubations. (R.T = room temperature). Dissolve first antibody stocks into 1X 4M, then use 1X TNB for the last two detection layers

Compound  Temp °C  Time (minutes)

0.) Gently remove rubber cement, place slides into R.T. 2X SSC, allow coverslip to fall off, then begin wash

1.) Wash in 2X SSC  R.T.  5

2.) Wash in 2X SSC  42  10

3.) Wash in 2X SSC R.T. 5

4.) Wash in 1X 4T R.T. 5

5.) Incubate in 1X 4M 37 30

6.) Wash in twice with 1X 4M R.T. 10

7.) Incubate FITC-Avidin (1ul/100ul) + Rhod A-DIG (.5ul/100ul) + Mouse A-DIG (1ul/100) in 1X 4M 37C for 30 min (Rhod A-Dig is optional)              Wash twice in 1X 4T R.T 10

8.) Wash once in 1X TNT R.T. 5

9.) Incubate in Biotin-A-avidin (.5ul/100ul) + Dig-A-Mouse (1ul/100ul) in 1X TNB 37C for 30 min              Wash 3x in 1X TNT R.T. 15

10.) Incubate in FITC Avidin (1ul/100ul) + Rhod A-DIG (2ul/100ul) in 1X TNB 37C for 30 min              Wash 3x in 1X TNT R.T. 15

11.) EtOH Series (70%-90%-100%) R.T. 15

12.) Add 12 ul Vectasheild cover with a 22 x 40 coverslip, squash gently

Solutions

2X SSC 
100 ml 20X SSC
900 ml ddH20
					
5X 4T
20X SSC		
0.25% Tween 20		

10X TNT
1.0 M Tris 
1.5 M NaCl
0.25% Tween 20
pH 7.5
								
5X 4M
16% Dry bovine milk (Sigma)
20X SSC

5X TNB
0.5 M Tris (pH 7.5)
0.75 M NaCl
2.5% Blocking reagent (Roche)

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