(based on Godt 1992, with Modifications by Dworkin. Also see White, R. 1998. Immunolabelling of Drosophila in Drosophila a practical approach. Roberts. DB ed. Oxford press)
Dissection: Dissect larvae in 1xPBS in depression slides. Remove fat body and salivary glands (optional). Transfer tissue (inverted head with wing discs) directly with forcepts into a tube or well of a micro-titer plate. Keep on ice until fixation. Use 500ul of solution per tube. Generally 10-12 "heads" per tube work.
Fixation: Fix 20 min. at room temperature (RT). Keep the tube/plate with tissue on a nutator throughout this, and all subsequent steps.
• 4% paraformaldehyde dissolved in 1x PBS with 50mM EGTA (from a 0.5M stock of EGTA - may require NaOH to dissolve).
This solution should be made in advance (in the fumehood, on a stir/heating plate), and stored in 1mL aliquots at -20 degrees celsius. Mix well before adding fixative to tissues. See recipe below for making the 4% paraformaldehyde.
• Be aware that 4% paraformaldehyde with EGTA may not work for all antibodies.
In that case, use 4% paraformaldehyde without EGTA.
Methanol: Transfer tissue directly with forceps into a basket with methanol in a micro-titer plate. 500ul per well for 3~5 min. at RT.
Note: Some antibodies show low binding activity with methanol fixed tissues.
• for storage at -20 degrees, remove methanol & add 100% ethanol after methanol fixation.
• Skip this step for alpha-CT antibody
Wash: Rinse with PBT, wash 4x15 min (or 3x20 minutes). with PBT.
• PBT = 1x PBS & 0.1% Tween-20. This is a good time to “clean-up” tissues post fixation (remove fat etc.)
• Make 50ml in a tube. Do not vortex PBT. Gently invert several times.
• Be aware that Tween-20 has a high viscosity, so pipette carefully.
Blocking: in 750~1000ul of PBTBS from 1 hour to overnight (O.N.) at 4 degrees.
• PBTBS = PBT + 0.1% Bovine serum albumin + 2% Goat serum.
• PBTBS can be kept for 2~3 days at 4 degrees.
• To make 3ml PBTBS :
60ul GS(100% solution) + 40ul BSA(7.5% solution) + 2900ul PBT
Do not vortex GS but BSA is vortex-able.
Note It is important that your blocking proteins DO NOT originate in the species in which the primary antibody was raised. This can mess up the specificity of the secondary antibody. Also some like to specifically use blocking serum from the same species as the secondary antibody was produced in. Generally not necessary though.
Incubate with primary antibody: in 1000ul of antibody w/ PBTBS overnight (O.N.) At 4 degrees Celsius. Some antibodies need to be pre-absorbed.
• dilute antibody with PBTBS & invert genetly or pipette to mix.
Wash: Rise with PBT; wash 4x 15min. RT.
Blocking: 60 minutes to O.N. In PBTBS at 4 degrees.
Incubate with Secondary antibody: 3hr to O.N. At 4 degrees Celsius. Secondary antibodies may also be pre-absorbed in PBTBS. For fluorescently labelled antibodies, long incubations tends to increase background levels. For Jackson laboratories antibodies dilutions of 1:400~1:600 work well.
• for imaginal disc secondary antibodies, use 1:1000 dilution.
Wash:As above
Staining (for immunochemistry): transfer disks to the depression slide with 500ul of DAB-PBT. Add 0.5microL 30% H2O2 (hydrogen peroxide). Watch as reaction progresses. When sufficiently dark, rinse 3~5 times in PBT to stop reaction. DAB-waste should be mixed with some bleach, allowed to sit for 5 minutes and then discarded in a DAB container. DAB is a carcinogen so be careful. Mount in 70% glycerol (prepared with a small crystal of phenol, to prevent growth), and seal with nail vanish.
• DAB-PBT: Dilute DAB (1 mg/mL stored in aliquots at -20 degrees C - keep in the dark) 1:3-1:4 with PBT.
Staining (for immunoflourescence): Mount in anti-fade (see below). Some seal in nail polish, but in my experience this can quench the fluorescence.
Pre-absorption step: Fix embryos as described in White (1998). Store in ethanol at -20 degrees C. Before using the embryos for pre-absorption, rinse X2, wash 3X5 minutes in PBT. In PBTBS, dilute antibody 1:10 (if possible), based on final concentration and amount of antibody to be used. Place diluted antibody 1:1 with embryos (i.e. equal volume of embryos and diluted antibody). Allow pre-absorption to occur for at least 1hr at room temperature, or O.N at 4 degrees Celsius. Dilute antibody to final concentration in PBTBS and use.
- 1.25g DABCO (sigma D-2522)
- Dissolved in 15mL PBS (1x). PBS should be at pH 7.4 (7.3-7.5 range)
- Add 35mL Ultra pure Glycerol
- Mix Gently for ~1-2 hours and store in 500 microL aliquots at -20 degrees C.
We also mix Hoechst into the anti-fade as a general nuclear marker. Mix 1 uL Hoechst:10000 microL anti-fade. Sometimes this is not bright enough so we do 1:1000, 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).
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, etc...
Also, since PF only lasts for ~6 months (before it starts producing by products like methanol), it may be worth making smaller amounts of this recipe (so scale it down to 25mL for instance).
- 100mL 1xPBS
- Pour 1xPBS into a conical flask containing 4g of paraformadehyde (powder).
- Cover with parafilm and take to fumehood.
- Place flask on the top of the hot plate with stirrer inside fumehood. Set heat control to 60-65 celsius with moderate stirring. Allow solution to warm up.
- When the para-formaldehyde is dissolved (solution is clear), switch off only the heat (keep stirrer on), and allow to cool.
- When it is cool, aliquot (0.5 mL/tube) and freeze.