CASFISH (Deng et al. PNAS | September 22, 2015 | vol. 112 | no. 38 | 11875)

1.     1.Cells cultured on 35-mm MatTek dishes or tissue sections were fixed at −20 °C for 20 min in a prechilled solution of methanol and acetic acid at a 1:1 ratio.

2.    2. Samples were washed three times (5 min each washing) with PBS with gentle shaking, followed by incubation for 15 min at 37 °C in Blocking/Reaction Buffer [20 mM Hepes (pH 7.5), 100 mM KCl, 5 mM MgCl2, freshly added 1 mM DTT, 5% (vol/vol) glycerol, 1% BSA, and 0.1% TWEEN-20].

3.   3. To assemble CASFISH probes, fluorescently labeled dCas9 protein (5–25 nM) was mixed with labeled or unlabeled sgRNA at molar ratio of 1:1 or 1:4, respectively, in blocking/reaction buffer and was incubated at room temperature for 10 min and stored on ice before the next step.

4.     4. 5 nM to 25 nM of fluorescent dCas9 protein or variant was used for CASFISH against target DNA elements.

5.    5. The assembled dCas9/ sgRNA was applied on preblocked cells and incubated for 5–30 min at 37 °C.

6.   6. he reaction was terminated by the removal of the dCas9/sgRNA solution and washing three times with blocking/reaction buffer.

Optional: CASFISH samples can be washed further to reduce background in buffer containing 20 mM Hepes (pH 7.5), 300 mM NaCl, 3 M urea, and 1.1% (vol/vol) Nonidet P-40. 

This protocol can also be used with Cas9-GFP protein.