Staining for proteins in Drosophila embryos

Common to the variations of the different staining protocols is the following basic scheme:
blocking with a protein solution
binding of primary antibody
washing off unspecifically bound antibody
detection of the primary antibody with a secondary antibody
washing off unspecifically bound antibody
colour reaction
mounting and microscopy
The specificity of the staining is determined by the ratio of signal and background. For each antibody an optimal dilution has to be determined (depends also on how the specimen was fixed). A high antibody concentration increases background, a low concentration reduces signal. For double/trible stainings it is important to know that secondary antibody are not absolutely specific. A goat-anti-rabbit IgG antibody may also weakly detect a mouse primary antibody, if this antibody was used in high concentration. Primary antibodies from guinea pig antibody are especially prone to be detected by other secondary antibodies, since these are purified against rabbit, mouse and rat cross-detection but not guinea-pig cross-detection.

DNA staining
fil. actin staining
membrane staining with concanavalinA

fixation, blocking and primary antibody

  • dechorinate embryos with bleach, fix in heptan/4% formaldehyde (PBS) for 20 min, pop with methanol
  • store embryos in methanol at -20°C
  • transfer embryos to PBT (methanol series is not necessary), wash 5 min
  • incubate for 1 h in PBT with 5% BSA or normal goat serum (blocking)
  • incubate for 2 h with the diluted antibody in PBT+0,1%BSA, or over night at 4°C
  • save the antibody solution, it may be reused several times, depending on the antibody. Some antibodies cannot be reused, such as a-pH3, other become better after multiple uses, such as a-En
  • rinse 3x with PBT
  • wash 4x15 min with PBT (500 µl)

detection with fluorescence

  • incubate 2 h (or over night at 4°C) with the diluted secundary antibody+0,1%BSA
  • rinse 3x
  • wash 4x15 min with PBT, or longer 2 h to reduce background
  • stain with DNA dye (5 min Hoechst, 1 h OliGree, Propidiumjodid, Toto-3, RNase at 50 µg/ml))
  • rinse 3x and wash with PBT (5 min)
  • mount in Aquapolymount, Mowiol/Dabco or other media

enhanced staining with ABC kit

  • the secondary antibody is biotinylated
  • incubate at a final concentration of 1:500 for 1-2h (overnight at 4°C)
  • rinse 3x and wash 4x15 min with PBT (without azide!)
  • 30 min prior to incubation mix A and B in PBT (no azide, each 1:100)
  • incubate 30-45 min with AB mix
  • rinse 3x and wash 3x15min with PBT (no azide)
  • set up staining, 500 µl PBT, 2µl 2% H2O2, start reaction with 500 µl DAB solution, reaction time may vary between a few seconds and minutes
  • stop the reaction by adding 10µl 40% NaN3 or washing with PBT
  • destroy/oxidise DAB with diluted bleach
  • attention: DAB is toxic, be extremely careful when pipeting DAB and decontaminate everything with diluted bleach

Background reduction:
The primary and secondary antibody may be preadsorbed (fixed + blocked embryos (lacking the antigen), incubate overnight, store with embryos at 4°C). Perform the staining with minimal volume of prim. and sec. antibody solution, e. g. 100 µl). Incubate inside the cap of an eppendorf cup.

double staining: 
stain first with the weaker antibody with only DAB. For the second reaction use the stronger antibody and develop with DAB and Ni2+ which result in a dark blue precipitate.

Ni2+:
stock: 10% NiCl2, final conentration 0,1%

H2O2: 
stock 20% (Merck), store at 4°C, final
            concentration should be higher than 0,003%
DAB: 
stock (2x) 0,4 mg/ml in PBS,  (Sigma D5637)
 3,3'-Diaminobenzidintetrahydrochlorid

filamentous actin (with Phalloidin)

  • fix embryos with 4-8% FA/PBS/Heptan for 20 min
  • remove fixative, add PBS
  • transfer embryos with a pasteur pipette from the interphase to a net
  • wash with PBT to remove all residual heptane
  • transfer dry embryos with a fine brush to a small petri dish with a double-sticky tape
  • cover with PBT
  • manually remove the vitelline membrane with a pulled out capillary
  • transfer embryos to PBT
  • stain for more than 30 min in labelled phallodine (1:1000, in PBT), may be used several times
  • stain for DNA
  • wash several times ( 3x 5min)
  • mount
PBT: PBS, 0,1%Tween20

azide: add 0,01% to protein solutions stored at 4°C

membrane staining with concanavalin A

  • fix and stain embryos as usual
  • stain with concanavalin A-Alexa together with the secondary antibody for 30 min to 1h (ditute 1:400, 2,5 µl in 1000 µl)
  • wash for 1h with four changes of PBT
  • stain for DNA
  • mount


DNA staining:

Hoechst: 

stock:  1 mg/ml, use 1:10000 (final 0,1 µg/ml)

Dapi:

stock: 0,4 mg/ml, use 1:500 (final 0,2 µg/ml). stain for less than 5 min, longer staining increases cytoplasmic background at least in embryos

oligreen  

(Molecular Probes, desicate at -20ºC): dilute 1:500 (stock, store at 4°C), stain at further 1:100 (strong) or 1:1000 (for confocal)

Propidiumjodid:  

stock: 1 mg/ml,  stain at 1:1000 (1µg/ml)

Toto-3:  

stock  1 mM (in DMF, dessicate at -20ºC), stain at 1:10000 (0,1 µM) von Molecular Probes

RNaseA treatment:

oligreen, Prop-jodid and Toto-3 bind ssDNA and dsDNA. To achieve nuclear staining incubate with 50 µg/ml RNaseA for at least 30 min prior to  or while staining with dyes.