Reagents and equipment

We've had several requests for part numbers for various reagents and the such for RNA FISH. Here's some info.

Reagents for fixation, wash, hybridization, anti-fade:

NF H2O, Ambion 4387936

Formamide, Ambion AM9342 (Aliquot and store at 4C; bring to room temperature before making buffers. Some people say this stuff goes bad really rapidly, but we haven't had a lot of trouble with that.)

20xSSC, Ambion AM9765

Dextran Sulfate, Sigma D8906-50G (store at 4C)

10xPBS, Ambion AM9624

Glucose oxidase, Sigma G2133-10KU (Dissolve 37mg in 10mL of 50mM Sodium Acetate, pH ~5.5 (diluted from 3M, pH 5.5) for a 3.7mg/mL stock. Make a bunch of aliquots and store at -20C. Each aliquot is good for ~20+ freeze/thaws.)

Catalase, Sigma C3515-10MG

Tris pH 8, Ambion AM9856

10% glucose solution (wherever)

(Only required for yeast) Zymolyase 100T (USBiological, L11042181), 2.5mg/ml in .06M Phosphate Buffer, pH 7.5

(For ethanol precipitation if you are coupling probes manually) 3M Sodium Acetate, pH 5.5, Ambion AM9740

Fixation solution:

5mL 10x PBS, 5mL 37% formaldehyde (100% formalin), 40mL NF H2O (can leave in hood at RT for months).

Wash buffer:

5mL 20x SSC, 5mL formamide, 40mL NF H2O (can leave in the hood at RT for months).

70% EtOH:

35mL 95% EtOH, 15mL NF H2O (lasts forever).

Hybridization buffer:

Add 1g dextran sulfate to 7mL NF H2O and mix by rotation for a while (could take several minutes to a half an hour). Then add 1mL formamide and 1mL 20x SSC and volume up to 10mL (volume by eye in a 15mL Falcon tube is fine). Store at -20C in 500µL aliquots; good for years.

Antifade buffer:

Note on catalase: vortex thoroughly before use. Try not to keep the bottle open very long because it is sometimes prone to contamination.

For the antifade buffer, use 850µl NF H2O, 100µl 20x SSC, 40µl 10% glucose, 10µl Tris. Use 900µl of this for a pre-wash before adding the actual antifade solution itself. To the remaining 100µl, add 1µl each of glucose oxidase and catalase; this is the antifade solution for use with Cy5/Quasar 670.

Buffer B (for fixing yeast cells):

1L stock: 1.2M sorbitol, 0.1M potassium phosphate dibasic, pH 7.5

300ml Nuclease-Free water

218g sorbitol powder (Mw=182.17g)

17.4g potassium phosphate dibasic powder

700ml Nuclease-Free water

Stuff for growing cells:

We often use chambered coverglass from Lab-tek. These are very handy because you can grow your cells directly in the chamber and then do all your fixation, hybridization, washing and even imaging right in the same well. Note that they also have a version 2 of this product, but we have honestly had less success with that product than with the original Lab-tek chambered coverglass. We usually get the two well chambers because you can slip a 18x18mm coverslip on top of you hybridization solution in order to spread out the solution and reduce evaporation, but the other sizes also work just fine.

Microscopy:

Pretty much any vanilla widefield microscope will work for single molecule RNA FISH. You want to have a good light source, a good camera, and a good objective. More on this soon.

Filter sets:

One of the most common questions is about the filter sets we use. Here they are: