For antibody staining the methanol was removed and replaced with PBT (PBT= PBS, 0.1% Triton X-100 and 0.1 % BSA). The embryos were re-hydrated by 4 X 15 minute washes in PBT and non-specific binding sites blocked with lOOpI PBT-NGS (950pl of PBT with 50pl of Normal Goat Serum from Sigma) for 30 min. The diluted primary antibody (Table 2.11) in PBT-NGS was added to the blocking solution to a final volume of 200pl. The embryos were incubated overnight at 4°C on a shaking platform. The next day the primary antibody was removed and the embryos washed with PBT for 4 x 15 minutes. Non-specific binding sites were blocked using lOOpI of PBT-NGS for 30 minutes. Another 99^1 of PBT-NGS were added together with 1pl of the secondary antibody to a final dilution of 1:200 (Table 2.12). The embryos were incubated with the secondary antibody for 2 hours at room temperature, followed by 3 x 15 minutes washes with PBT. If the antibody was fluorescently labelled the tube with embryos was covered with aluminium foil during the secondary antibody incubation and all the subsequent steps. After the last removal of the wash solution one drop of FluoroGuard Antifade Reagent (BioRad), a glycerol-based mounting medium containing anti-fading agent, was added and the tube placed at -20°C overnight to clear, i.e. equilibrate with glycerol. If the secondary antibody was HRP-conjugated the staining was developed using DAB, a colour reagent for visualisation (see 2.6.2).
2.6.1 Pre-absorption of primary antibodies
In the case that the primary antibody is rabbit polyclonal, it can be pre absorbed overnight to remove non-specific binding proteins. Fixed wild type embryos from an overnight collection were used in order for embryos from all developmental stages to be present. The primary antibody was added in the appropriate dilution, usually lOx more concentrated than the antibody concentration needed for the staining, in a PBT-NGS solution of final volume of lOO^il, and incubated overnight.
2.6.2 DAB staining
The secondary antibodies that are HRP-conjugated need to be developed with a colour reaction. After the final wash, the embryos were placed in a glass petri-dish (Agar) containing 1 ml of ddHaO and 1 tablet of DAB (tablets, 3,3'- diaminobenzidine) and one tablet of hydrogen peroxide (kit from Sigma). The embryos were left in this solution till the colour develops. The reaction was then stopped by several washes with PBS. The embryos were then placed in a microcentrifuge tube with 80% glycerol in PBS and left at room temperature to clear.
2.6.3 Mounting
The embryos were mounted on slides, either in 80% glycerol in PBS or FluoroGuard for colour or fluorescent labelling respectively. In both cases embryos were removed from the microcentrifuge tubes with the mounting medium using a glass micro-pipette and placed in the middle of a glass slide. If there was not enough mounting medium then more was added, in an approximate proportion of 30^x1 for 20 embryos. A cover-slip was then put on side of the embryos and another cover-slip placed on top of the other two covering the embryos by making a bridge to avoid squashing them. The fluorescently labelled embryos were stored at -20°C and DAB stained ones at room temperature.
2.6.4 Actin staining
Embryos were collected, dechorionated, fixed and devitellinised (see 2.4, 2.5.4). Ethanol was removed from the embryos by washing 3 x 1 5 minutes in PBT (PBS, 0.1% Triton X-100, 0.1% BSA). Non-specific binding sites were blocked in lOO^il of PBT-NGS (950pl of PBT and 50pl of normal goat serum) for 30 minutes. After that time another 80pl of PBS-NGS was added together with 20pil of TRITC-Phalloidin (phalloidin-tetramethylrhodamine conjugate from Sigma) prepared as described (see 2.9.3). The tube containing the embryos was covered with aluminium foil to avoid photo-bleaching of the fluorescently labelled substrate. The embryos were incubated at room temperature for 20 minutes and
then washed with PBT 3 x 1 0 minutes. After the final removal of PBT a drop of FluroGuard Antifade Reagent (BioRad), was added. The embryos were then left to clear in the medium overnight at -20°C.
2.6.5 Simultaneous actin and antibody staining
Staining of embryos with antibody and phalloidin simultaneously was carried out by the actin fixation and devitillinisation protocol (see 2.5.4), followed by antibody staining (see 2.6) and subsequent addition of phalloidin (see 2.6.4) prior to the addition of the mounting medium (see 2.6.3).
2.6.6 Actin and non-muscie myosin ii antibody staining
Dechorionated embryos were suspended in 4 ml PBS in a glass scinitillation vial, another 4 ml of the fix solution (2 parts PBS and 2 parts 16% ultra pure and methanol free formaldehyde) was gradually added while the vial was gently vortexed for 45 seconds. 4 ml of heptane was then added and the embryos agitated vigorously for 23 minutes. The reaction was then stopped and the embryos washed several times with PBS. The embryos were placed on double sided sticky tape on a slide with a drop of PBS to avoid drying and the vitelline membrane removed using a tungsten needle (Agar).
2.6.7 Amplification methods for antibody staining 2.6.7.1 Enhancement of colour substrate
Weak antibody colour staining is enhanced using an amplification step provided in the ABC Elite kit (Vector laboratories) according to the manufacturers' instructions. The standard antibody staining method was followed until the end of primary antibody incubation. Subsequently non-specific binding sites were blocked for 30 minutes in 10Ofil of horse serum provided in the kit. The blocking solution was prepared by adding 1 pil of blocking serum to 99pil of PBT. The blocking solution is then removed and the universal secondary biotinylated antibody added. The secondary antibody solution was made up of 192^1 of PBT, 4pil of blocking serum, and 4\i\ of the secondary antibody. The embryos were incubated in the secondary antibody solution for 2 hours at room
temperature. They were then rinsed in PBT and washed 4 x 1 0 minutes in PTW (PBS with 0.1% Tween-20 (polyoxyethylene (20) sorbital monolaurate from Sigma). After the washes the embryos were incubated for 30 minutes at the ABC mix solution for the amplification reaction to proceed. The ABC reaction mix is composed of 192pl of PTW, 4pil of solution A and 4^1 of solution B provided by the kit. The reaction mixture should be prepared, mixed immediately and allowed to stand for 30 minutes before being added to the embryos. After this incubation the embryos were washed 4 x 1 0 minutes in PTW followed by a colour development reaction (see 2.6.2)
[The ABC Elite kit contains normal blocking serum, biotinylated, affinity purified anti-immunoglobin, reagent A, which is Avid in DH, and reagent B, which is the enzyme].
2.6.7.2 The TSA Fluorescence System - Tyramide signal
amplification kit (MEN- Perkin & Elmer Life Sciences Inc)
To enhance fluorescent staining, a HRP-conjugated secondary antibody was used instead of a fluorescently conjugated one. The secondary antibody and incubation was carried out by following the standard protocol described (see 2.6). Tyramide-fluorescein at a 1/50 dilution in the buffer provided by the kit was then added and the reaction incubated for maximum of 10 minutes, e.g. for anti twist incubation lasts only 4-5 minutes, othenA/ise the signal is very strong. The embryos were then washed and processed as in the standard protocol described (see 2.6 and 2.6.3). Since the tyramide is fluorescently conjugated the tube containing the embryos should be wrapped in aluminium foil to avoid photo- bleaching.
[The TSA kit contains streptavidin-horseradish peroxidase, blocking reagent, amplification diluent and fluorescein-labelled tyramide. Tyramide is diluted in DMSO (dimethyl sulphoxide from Sigma)].
In both the amplification protocols, extended washes after the primary antibody binding are required to avoid non-specific amplification and high background signals.
2.6.8 Primary antibodies used for staining embryos
Table 2.11
Name Species Source
Additional requirements Concentration Anti-Engrailed mouse monoclonal DSHB 1/5 22C10 mouse monoclonal DSHB 1/20 Anti-Crumbs mouse
monoclonal Arno Muller
with ABC Kit 1/50 Anti-non-muscle myosin II rabbit
polyclonal Christine Field
1/700 Anti-p- galactosidase rabbit polyclonal Cappel, ICN biochemicals 1/100 Anti-p-subunit of integrin mouse
monoclonal Nick Brown
1/10
Anti-Twist
rabbit
polyclonal Siegfried Roth
Preabsorbed, with TSA kit
1/1000
2.6.9 Secondary antibodies used for staining embryos and imaginai discs
Table 2.12
Name Species Conjugated Source Concentration
FITC-anti- mouse IgG donkey fluorescein- conjugated Jackson Laboratories 1/200 TRITC-anti- mouse IgG donkey rhodamine- conjugated Jackson Laboratories 1/200 FITC-anti- rabbit-IgG donkey fluorescein- conjugated Jackson Laboratories 1/200 TRITC-anti- rabbit-IgG donkey rhodamine- conjugated Jackson Laboratories 1/200 HRP-anti- mouse-IgG goat peroxidase- conjugated DAKO 1/200 HRP-anti- rabbit-IgG goat peroxidase- conjugated DAKO 1/200 2.7 Cuticle preparations
The embryos were allowed to develop to late stages in order for the cuticle to be formed, dechorionated and incubated in 9 parts of 85% lactic acid (Fluka) and 1 part 96% ethanol (BDH) for 24-36 hours at 50°C. The tubes that contained the embryos were gently shaken every few hours. Then the embryos
were transferred to slides (BDH) with glass micro-pipettes, and 50 x 22 mm coverslips (BDH) were placed on top. The slides were ieft on a hot plate at 60°C for 24 hours for the lactic acid to dry and for the coverslip to stick to the slides. The cuticles were photographed in a dark field.