1.6.2. Dissection and fixation of ovarioles

1. Dissect the ovaries from adult queens or workers (see the BEEBOOK paper on anatomy and dissection (Carreck et al., 2013)).

2. Transfer these to a dish containing honey bee culture medium (recipe and preparation steps as described in Rachinsky and Hartfelder, 1998) or a commercial insect culture medium (e.g. TC-100).

3. Individualize the ovarioles and, with the aid of fine watchmakers forceps, manually remove the tracheae and the peritoneal sheath covering each ovariole.

4. Transfer batches of individualized clean ovarioles into a 4-well plate containing sufficient (around 0.5 ml) Stabilization Buffer (see 1.6.1., step 2.) to cover the ovarioles.

5. Keep for 20 min at room temperature (RT) under shaking movement.

6. Fix the ovarioles in cold (-20°C) pure methanol for 10 min at 4°C.

Note, the most common fixative used in immunolabelling protocols is 4% paraformaldehyde (PFA) in PBS, but for microtubules, a better result is obtained with methanol fixation following treatment with Stabilization Buffer.

7. Rinse in PBS for 30 min at RT with shaking.

8. Permeabilize in PBS-T (PBS 10 mM, Triton X-100, 0.1 %) for 30 min at RT with shaking.

9. Block unspecific binding sites by incubating the ovarioles in PBS-T supplemented with 10 % non-immune serum for 20 min at RT with shaking.

The non-immune serum must correspond to a serum of the organism used as source for generating the secondary antibody, e.g., if using a FITC-conjugated goat-anti-mouse-IgG  antibody, use a non-immune goat serum for blocking.

10. Rinse in PBS-T for 2x 15 min at RT with shaking.

11. Incubate for 3 h at RT or at 4°C overnight in a monoclonal antibody raised against α-, β- or γ-tubulin, diluted in PBS-T under shaking.

When using the monoclonal antibody raised against βI+II-tubulin (Sigma-Aldrich, T8538) a dilution of 1:200 in PBS-T may give good results, but such dilutions must be determined empirically by serial dilution experiments for each antibody and tissue.

12. Rinse in PBS-T for 2 x 30 min at RT with shaking.

13. Remove PBS-T.

14. Leave ovarioles for 1 h in a fluorescence-conjugated secondary antibody diluted in PBS-T in darkness at RT under shaking.

In the case of a FITC-conjugated goat-anti-mouse IgG (Sigma-Aldrich), a dilution of 1:400 in PBS-T is sufficient to obtain a good signal to background ratio.

15. Rinse the ovarioles in PBS-T for 2 x 30 min in darkness at RT with shaking.

16. After this wash step one can add TRITC-conjugated phalloidin to label filamentous actin, and/or DAPI (4',6-diamidino-2-phenylindole) or another reagent intercalating with dsDNA to counterstain nuclei.

17. Rinse the ovarioles in PBS-T for 2 x 30 min in darkness at RT with shaking.

18. Rinse briefly in ddH2O.

19. Transfer ovarioles to microscope slides and embed in glycerol/n-propyl gallate mounting medium:

19.1. Glycerol 90 %,

   19.2. N-propyl gallate 3 % in PBS,
   19.3. Supplemented with sodium azide 0.01 %.

N-propyl gallate is an anti-fade reagent used in fluorescence microscopy to reduce photobleaching; alternatively, you may use commercial antifade reagents (e.g. Vectashield).

20. Coverslip.

21. Seal coverslip edges with nail polish.

21. Store slides in dark.

The whole-mount ovariole preparations can be analysed by conventional epifluorescence microscopy or by means of a laser confocal system.

As in any immunolabelling protocol, a negative control staining is mandatory. In the case of commercial antibodies this can be done by substituting the primary antibody for PBS-T. When using an antibody prepared in the proper laboratory or by a colleague, it is even better to use at this step a pre-immune serum, i.e. serum drawn from the respective animal (usually rabbit) prior to immunization.

User safety: Sodium azide is highly toxic. Appropriate ventilation (laboratory chemical hood) and personal protective equipment (such as gloves) must be used to minimize exposure.

The BEEBOOK