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Recipes for stock solutions and other reagents for the lab.

Phosphate Buffered Saline (PBS)

Use: PBS is used as a general saline buffer for dissection of imaginal discs (and other tissues) for RNA extraction, antibody stainings, examining GFP reporters in discs etc.. Also used as component for other solution (i.e. PBS/glycerol) for mounting.

Note: For routine use of PBS (for mountants for instance) homemade 10X PBS is fine. For applications for RNA extraction, we typically by PBS that is certified RNase free.

There are many variants of PBS recipes. This one for 10X PBS is from the Drosophila protocols book (CSHL Press, 2000) page 656. It Makes 1L of 10X PBS.

  • 80g NaCl
  • 2g KCl
  • 14.4 g Na2HPO4
  • 2.4 g KH2PO4
  • 800 mL milliQ autoclaved water.
  1. Combine all components in the 800mL H2O and stir to dissolve.
  2. Adjust pH to 7.2
  3. Adjust volume to 1 litre (1000 mL) with autoclaved milliQ water.
  4. Dispense in convenient volumes and sterilize by autoclaving. Store at room temperature.

PBT (with Tween-20 as the detergent)

Use: PBS buffer with detergent. Detergent increases permeability of cell membrane (breaks holes in cell membrane), to allow passage of small molecules (like antibodies, GFP, DAPI, Hoechst, Phalloidin, etc..).

PBT (Triton-X)

Use: PBS buffer with detergent. Detergent increases permeability of cell membrane (breaks holes in cell membrane), to allow passage of small molecules (like antibodies).

PBTBS (PBT with blocking solution)

Use:

Glycerol/PBS mounting solution for adult wings.

Use: This is a quick and useful mountant for adult Drosophila wings. Wings mounted in this are generally good enough for wing size (area), morphometrics and trichome counts. Mounted wings stored at room temperature start to degrade after 9-12 months.

  1. Mix a solution that is 70% Glycerol and 30% 1X PBS (V/V), add a few crystals of phenol to solution. Mix on nutator for 1-2 hours, then aliquot. 1b. For 40mL of the solution this is 28mL of glycerol with 12mL of PBS. You may wish to centrifuge the glycerol first unless it is molecular grade.

4% Paraformaldehyde

Use: Fixative for immunofluorescence (antibody staining).

Notes:

  • Wear gloves, lab coat and mask (PF is not good for you). Do all of it in fumehood. Paraformaldyhe sticks to everything,even after washings, so use dedicated flasks, weighing boats, stirring rods, etc...

  • Since PF only lasts for ~6 months (even frozeb) before it starts producing byproducts that may cause problems, it may be worth making smaller amounts of this recipe (so scale it down to 25mL for instance). This will depend on needs in the lab.

  1. Measure 100mL 1xPBS
  2. Pour 1xPBS into a conical flask containing 4g of paraformadehyde (powder). Note: I move small scale to fumehood for weighing.
  3. Cover with parafilm and take to fumehood.
  4. Place flask on the top of the hot plate with stirrer inside fumehood (or manually mix flask every few minutes). Set heat control to 60-65 celsius with moderate stirring. Allow solution to warm up.
  5. When the para-formaldehyde is dissolved (solution is clear), switch off only the heat (keep stirrer on), and allow to cool.
  6. When it is cool, aliquot (0.5 mL/tube) and store at -20C.

anti-fade mountant for fluorescent samples: From Goldstein (pp383): (with and without Hoechst)

use: This is used as a mountant for samples where flourescence (i.e. GFP, antibodies conugated with fluorescent probe, DAPI, Hoechst) is going to be examined. Anti-fade prolongs the functional life of the samples.

DABCO (1,4-diazabicyclo[2.2.2]octane) based anti-fade mountant.

Note: This makes 50mL of antifade. Depending on lab uses in the next 6-12 months you may wish to decrease (or increase) the amount you make. Ask your fellow researchers in the lab!

  1. 1.25g DABCO (sigma D-2522) (2.5%)
  2. Dissolved in 15mL PBS (1x). PBS should be at pH 7.4 (7.3-7.5 range)
  3. Add 35mL Ultra pure Glycerol
  4. Mix Gently for ~1-2 hours and store in 500 microL aliquots at -20 degrees C.
  • Some protocols use 1% DABCO, not 2.5% I have not tested this. Also some make the mountant in 90% Glycerol/ 10% PBS. I have not tried this either.
  • An alternative to DABCO is to use n-propyl gallate (usually at 4%), in the glycerol/PBS mountant. I have not tried this.
  • Read here and here for more information.

Adding Hoechst or DAPI to anti-fade mountant as a nuclear co-stain.

  • For more information on using Hoechst or DAPI in our lab see here.
  • We also mix Hoechst into the anti-fade as a general nuclear marker. Mix Hoechst 33342 to a final concentration of 1 ug/mL. Exact amount depends on stock concentration. Sometimes this is not bright enough so you can go as high as a final concentration of 10 ug/mL (but try 2, 5 before making jump to 10) depending on application.
  • Anti-fade with Hoechst is stable for ~1 month in the freezer.
  • Store Hoescht stock solution at -20C for long term, and some at 4C for short term use (stable for ~6 months).

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