• 12. Hold the salivary glands at the common duct with tweezers.Transfer the glands to a drop of fixing solution on a siliconizedcoverslip. 
  
  

• 13. Incubate the glands for 10-20 min, occasionally stirring with the tip of the tweezers to ensure homogeneous fixation. 
  The fixation time is an important parameter and needs to be adjusted for every antigen tested. Aim for the shortest time necessary to visualize the signal, as extended fixation times will result in difficulties with spreading the chromosomes. Unlike the sole treatment with acids,e.g., Dissection of Larval Salivary Glands and Polytene Chromosome Preparation (Kennison 2008), the inclusion of formaldehyde in the fixing solution keeps the chromosome sticky, making it more difficult to obtain preparations with well-spread chromosome arms. 
  

Preparing Chromosome Squashes

• 14. Take up the coverslip with a poly-L-lysine-treated slide. 
  
  

• 15. Tap the coverslip with the eraser end of a pencil until the cells are broken up. 
  This can be monitored best when done against a black background. 
  
  

• 16. Move the eraser end of the pencil over the coverslip to spread the chromosomes. To avoid extensive movement of the coverslip, hold down the coverslip with the tip of a finger (use latex gloves to prevent acid burns). Do not continue to spread the specimen if the coverslip sticks to the slide, as this will result in shearing of the chromosomes. 
  The chromosomes are very brittle at this point of the process. The extent of spreading depends on the constitution and size of the chromosomes, as well as on the time of fixation. 
  
  

• 17. Invert the slide, and place it over one layer of filter paper. Apply firm pressure with the thumb; the chromosomes become flattened and pressed to the slide. 
  The squashing apparatus (Fig. 1) can substantially ease this step because it allows more firm and homogeneous pressure on the chromosomes without the risk of moving the coverslip. 
  
  

• 18. Examine the preparation by phase-contrast microscopy. Use only preparations with well-spread chromosome arms showing high-contrastbanding patterns. 
  
  

• 19. Mark the position of the coverslip with a diamond pen. 
  
  

• 20. Freeze the slide in liquid nitrogen. 
  
  

• 21. Remove coverslip with a razor blade (wear appropriate eyeprotection). 
  
  

• 22. Immediately dip the slide into PBS, and wash it for 15 min with gentle shaking. Repeat this wash once in fresh PBS. 
  
  

• 23. Proceed with the immunostaining as described below. Alternatively, keep the slide (for up to 1 wk) in 100% methanol or in 50% (w/v) ammonium sulfate at 4°C. 
  Certain antigens might be affected by exposure to methanol. 
  

Immunostaining

• 24. Wash stored slides twice in PBS (10 min each wash). 
  
  

• 25. Wash slides once in PBS containing 1% Triton X-100 for 10 min. 
  
  

• 26. Incubate slides in a slide jar containing blocking solution for 1 h at room temperature or overnight at 4°C with gentle shaking. Drain off excess solution. 
  
  

• 27. Add 40 µL of diluted affinity-purified primary antibody to each slide, and cover with a coverslip. Incubate the slides for 1 h at roomtemperature. 
  
  

• 28. Incubate the slides overnight at 4°C in a humid chamber. 
  
  

• 29. Rinse the slides in PBS to remove the coverslips, and place them in a rack. Wash the slides three times in blocking solution (5 min each wash) with thorough shaking. 
  
  

• 30. Probe the slides with a secondary antibody detection system using one of the three methods below. 
  If only weak signals are observed using enzyme-coupled secondaryantibodies (Steps 30.i-30.vi), amplification with the biotin-avidinsystem might be helpful (Steps 30.vii-30.xiii). Secondary antibodiestagged with a fluorochrome (Steps 30.xiv-30.xvii) can be used to speed up the detection procedure, as well as for double-labelingexperiments. 
  

  • To immunostain using enzyme-coupled secondary antibodies: 
    

  • i. Rinse slides in PBS and remove excess solution. 
    
    

  • ii. Add 40 µL of secondary antibody (e.g., anti-rabbit IgG [Fc] HRP conjugate, 1:100 dilution) to each slide, and cover with a coverslip. Incubate for 1 h at room temperature in a humid chamber. 
    Often, commercially available secondary antibody preparations show a high affinity for Drosophila nuclear proteins, resulting in some excellent signals on the chromosomes. As such, a correspondingcontrol experiment should always be included. 
    
    

  • iii. Rinse slides in PBS and place in rack. Wash slides in wash solution 1 for 15 min and then in wash solution 2 for 15 min. Shake rack thoroughly during the washing procedure. 
    
    

  • iv. Rinse slides in PBS. Add 100 µL of a solution containing 0.5 mg/mL DAB and 0.01% H₂O₂. 
    
    

  • v. To prevent overstaining, follow the appearance of the signals under the microscope using bright-field optics. Stop the reaction by dipping the slides in PBS. Wash in PBS for 10 min. 
    
    

  • vi. Counterstain with Giemsa (see Steps 30-33). 
    

  • To immunostain using biotin-conjugated secondary antibodies and enzymatic detection with the avidin/biotin system: 
    

  • vii. To each slide, add 40 µL of secondary antibody conjugated to biotin, and cover with a coverslip. Incubate for 1 h at roomtemperature in a humid chamber. 
    
    

  • viii. During secondary antibody incubation, mix the biotin solution and the avidin-HRP solution to allow the formation of complexes. Mix 40 µL of solution A and 40 µL of solution B (from the VECTASTAIN Elite ABC kit) in 1 mL of PBS containing 0.1% BSA for 10 min at room temperature. 
    
    

  • ix. After incubation with secondary antibodies, rinse slides twice in PBS containing 0.1% BSA (10 min each). Remove excess liquid. 
    
    

  • x. Place 50 µL of the biotin-avidin-HRP mix on the preparation and cover with a coverslip. Incubate the slides for 40 min at room temperature in a humid chamber. 
    
    

  • xi. Wash slides in wash solution 1 for 15 min and then in washsolution 2 for 15 min. 
    
    

  • xii. Rinse slides in PBS. Add 100 µL of a solution containing 0.5 mg/mL DAB and 0.01% H₂O₂. 
    
    

  • xiii. To prevent overstaining, follow the appearance of the signalsunder the microscope using bright-field optics. Stop the reaction by dipping slides in PBS. Wash in PBS for 10 min. 
    

  • To immunostain using fluorescence-coupled secondary antibodies: 
    

  • xiv. Rinse slides in PBS and remove excess solution. 
    After this point, perform all steps under dimmed-light conditions.During incubations, wrap the slide jars in aluminum foil to minimize bleaching of fluorochromes. 
    
    

  • xv. Add 40 µL of diluted fluorescence-conjugated secondary antibody solution to the slide, and cover with a coverslip. Incubate for 1 h atroom temperature in a humid chamber. 
    For double-labeling experiments, both secondary antibodies of the appropriate type can be applied simultaneously during this step. 
    
    

  • xvi. Rinse slides in PBS and place in a rack. Wash slides in wash solution 1 for 15 min and then in wash solution 2 for 15 min. 
    
    

  • xvii. Store slides in PBS at 4°C in the dark or proceed to counterstain with Hoechst 33258 (see Steps 35-37). 
    

Giemsa Staining

Chromosomes with HRP signals should be counterstained with Giemsa.

• 31. Prepare a 1:130 dilution of Giemsa in 10 mM sodium phosphatebuffer (pH 6.8). 
  
  

• 32. Stain slides for 30 sec to 1 min. Rinse slides by dipping several times into H₂O. 
  
  

• 33. Mount in 99.5% glycerol with a coverslip. Immediately examinethe slides under the microscope. 
  Giemsa stain will fade within a few hours. However, chromosomes can be washed in PBS and restained. 
  
  

• 34. For storage, slides can be frozen at –20°C. Entellan (EMD) can be used as a permanent mounting solution. 
  

Staining with Hoechst 33258

Chromosomes with fluorescent signals can be counterstained withHoechst 33258. However, banding patterns produced by the fluorescent DNA stains are often difficult to compare with the published polytene chromosome-banding patterns. It is often more convenient to simply use phase-contrast to correlate the fluorescent signal with the corresponding chromosome bands.

• 35. Stain slides in Hoechst staining solution for 5 min. 
  
  

• 36. Mount preparations with fluorescent signals in Mowiol-DABCOstock solution. 
  
  

• 37. Examine the slides using a fluorescence microscope. 
  

TROUBLESHOOTING

Problem: Background staining is high.

[Steps 30, 33, or 37]

Solution: The concentration of NaCl in wash solution 2 can be raised to 500 mM.

Problem: HRP staining is weak.

[Step 30]

Solution: Silver treatment of the preparation turns the brown color of a DAB signal to black, thus substantially improving the contrast, which is very convenient when black-and-white photography is used for documentation. Use the Sigma FAST DAB with metal enhancer (Sigma D0426), following the supplier’s instructions, but shorten the metal amplification step to ~1 min.

Problem: In Giemsa-stained slides, staining of chromosome bandsappears too weak under bright-field optics.

[Step 33]

Solution: Repeat Giemsa staining starting with Step 32.

DISCUSSION

Immunostaining of polytene chromosomes is most often used formapping distributions of chromosome-associated proteins, identifyingand characterizing cis-regulatory DNA elements bound by particularproteins, and mapping functional protein domains necessary forchromosomal binding or other activities, such as the interaction with other partner proteins. For example, a transgene construct containing a cis-regulatory DNA element may create a new protein-binding site at the integration site on a chromosome (Fig. 2 ).

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Figure 2. Distribution of the Polycomb protein at section 43-49 of the second chromosome visualized with enzyme-conjugated (HRP) secondary antibodies. (Top) Chromosome with a transgene construct containing a Polycomb-binding site (PRE) at 44E (arrowhead); (bottom) wild-type chromosome.