Mapping Protein Distributions on Polytene Chromosomes by Immunostaining-1

INTRODUCTION

The formidable size and structure of polytene chromosomes allowmapping of chromosomal protein distributions at very high resolution.This protocol describes the preparation of polytene chromosomes from Drosophila larvae, immunostaining of the chromosomes with a protein of interest, and counterstaining with Giemsa and Hoechst.

RELATED INFORMATION

Technical modifications incorporated into this protocol that have enabled more reproducible patterns of banding in immunostainedchromosomes were initially described by Silver and Elgin (1976). For further details on dissecting larval salivary glands, see Dissection of Larval Salivary Glands and Polytene Chromosome Preparation(Kennison 2008).

MATERIALS

Reagents

 Ammonium sulfate (50% [w/v]) (optional, see Step 23)

Antibodies, primary

Affinity-purified primary antibodies are diluted in PBS containing 1% BSA. The dilutions must be adjusted for each individual primaryantibody. Typical dilutions for rabbit polyclonal antibodies range between 1:50 and 1:500. For double-labeling experiments, use primary antibodies raised in two different species.

Antibodies, secondary

Secondary antibodies are diluted in PBS containing 2% normal serum, obtained from the same species as the secondary antibody. Depending on the method employed, use anti-rabbit IgG (Fc) horseradishperoxidase (HRP) conjugate (1:100 dilution), a biotin-conjugatedsecondary antibody, or a fluorescence-conjugated secondary antibody.For double-labeling experiments, use fluorescence-conjugatedsecondary antibodies raised to the corresponding type of primaryantibody. The dilution factor for each antibody needs to bedetermined experimentally.

Blocking solution (a small spoonful of nonfat dry milk in 40 mL PBS)

Bovine serum albumin (BSA)

 3,3''-diaminobenzidine tetrahydrochloride (DAB; Sigma D5637)

Detergent (optional, see Step 2)

Drosophila (see Steps 7-9)

 Entellan (EMD) (optional, see Step 33)

Ethanol (95%)

 Fixing solution

 Giemsa (Merck 109204)

Glycerol (99.5%)

 H₂O₂ (Merck 107210)

 Hoechst staining solution

 Liquid nitrogen

 Methanol (optional, see Step 23)

 Mowiol-DABCO stock solution

 Nutrient-rich fly medium

 Phosphate-buffered saline (PBS; pH 7.5)

 Poly-L-lysine solution (0.1% [w/v] in H₂O; Sigma P8920)

 Sodium phosphate buffer (10 mM, pH 6.8)

 Triton X-100

VECTASTAIN Elite ABC Kit (Vector Laboratories PK 6100) (optional, see Step 30)

 Wash solution 1

 Wash solution 2

Equipment

Aluminum foil

Diamond pen

Eye protection (see Step 21)

Filter paper (e.g., Whatman 3MM)

Humid chamber for slide incubation

Latex gloves

Microscope, dissecting

Microscope, phase-contrast

Pencil with eraser end

Rack for holding slides

Razor blade

Shaking platform

Siliconized coverslips (Corning or equivalent; 22 x 22 mm)

Slides

Slide jars

Squashing apparatus (optional, see Step 17)

For extended chromosome-spreading sessions, use the custom-madesquashing apparatus (Fig. 1 ).

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Figure 1. Schematic diagram of the squashing apparatus. This apparatus eases the effort when squashing chromosomes over an extended period, and produces more homogeneous pressure over the entire coverslip. A small block made of polyvinyl chloride (2.5 x 2.5 x 1.5 cm) is attached to a flexible Teflon ribbon as shown. The block should hang ~2-3 mm above the plane of the holding block (also made of polyvinyl chloride). A piece of filter paper (i.e., Whatman 3MM) and the slide with the coverslip are then positioned on the holding block and held in place by the suspended block.

Tweezers

METHOD

Preparation of Poly-L-Lysine-Coated Slides

• 1. Place 100-200 slides in racks. 
  
  

• 2. (Optional) Wash slides for 2 h in a strong detergent. 
  In general, high-quality slides do not require any pretreatment with detergent. However, in some cases, pretreatment with a strong detergent may be necessary to ensure a homogeneous wetting of the surface by the poly-L-lysine solution in Step 5. 
  
  

• 3. Wash for 2 h in running tap water. Rinse twice in H₂O. 
  
  

• 4. Dip slides twice in 95% ethanol. Air-dry. 
  
  

• 5. Dip slides into the poly-L-lysine solution. 
  The solution should wet the glass surface uniformly and stay on the slides. 
  
  

• 6. Air-dry the slides, and store them at 4°C. 
  

Raising Third-Instar Larvae

The quality of the chromosomes depends critically on the state of nutrition and well-being of the larvae. This is particularly important to consider in cases where the genetic background (e.g., unhealthy mutants or certain transgenic lines) places a heavy toll on the development of the larvae.

• 7. Raise flies in 175-mL bottles containing nutrient-rich fly medium. 
  
  

• 8. Allow the flies to lay eggs just to the point where larvae will hatch under uncrowded conditions (i.e., fewer than 100 larvae in a 175-mL bottle). Let the larvae develop at 18°C. 
  
  

• 9. For salivary gland preparations, use third-instar larvae that are still crawling and have not yet started to pupate. 
  

Dissecting Larval Salivary Glands and Fixing Chromosomes

• 10. For each slide, remove two larvae from the bottle, and wash them in PBS. 
  
  

• 11. Keep the larvae in PBS, and dissect out the two pairs of salivary glands from each larva using tweezers. Carefully remove most of the attached white fat-body cells without damaging or separating the salivary glands. 
  For further details on dissecting larval salivary glands, see Dissection of Larval Salivary Glands and Polytene Chromosome Preparation (Kennison 2008).