Make fresh daily:

• 100ul 10X Buffer A
• 100 ul 10% Triton X
• 800 ul Water

• 100 ul 10X Buffer A
• 100 ul 10% Triton X-100
• 231 ul 16% Formaldehyde stock (use EM grade)
• 569 ul Water

• 500 ul glacial acetic acid (use from special bottle)
• 500 ul Water

Stock solutions

• 15 mM Tris-HCl pH 7.4
• 60 mM KCl
• 15 mM NaCl
• 1.5 mM Spermine
• 1.5 mM spermidine

Acetic Acid is obtained from Sigma Catalog # A6283 - 100 ml size

Preparing the glands.

1.  Dissect larvae in 100 ul of Solution A. (Use late 3rd instar larvae from a healthy, uncrowded bottle.)
2. Move salivary glands to 50 ul of Solution B for 30 seconds.
3. Dip glands in Solution G for a few seconds (do not let go of glands if possible, just dip in and out).
4. Move glands to a siliconized coverslip with 23 ul of Solution G on it.
5. Let set for 3-4 minutes.
6. Place the cover slip near the edge of the bench and place a slide on the cover slip, approaching the coverslip from an angle (/-).  Spread the chromosomes by first tapping on coverslip directly over glands with a needle probe. Then, again with the probe, rub in back and forth motion letting the coverslip move a little bit. Then hold the coverslip with one hand and again go over the coverlsip in a gentle scratching motion either back and forth or circular or what ever design you like. Look at spreads to determine if you have good spreads.
7. Very carefully (using forceps) put good slides into liquid nitrogen (lower them in very slowly or they will crack), making sure the slide will be completely covered! Leave for 2 to 5 minutes.
8. Take out with forceps, blow on the coverslip and quickly flick off coverslip with a razor blade, and with a diamond pencil quickly mark were coverslip was. Then put slide into coplin jar with 95% EtOH (EtOH should cover slide).
9. If storing slides overnight, put coplin jar of 95% EtOH and slides into refrigerator.
10. Store them for 24 hrs maximum.

Staining the chromosomes for immunofluorescence analysis.

Need TBS (start with 1 liter) : 150 mM NaCl, 10 mM Tris pH 7.0-7.5

1. Rehydrating Samples

• Pour off the EtOH.
• Pour TBS in with slides. Leave for 5-10 minutes.
• Pour off TBS. Put in new TBS and leave for 5-10 minutes.

• The blocking solution is 10% Fetal Bovine Serum (with 0.05% Sodium Azide) in TBS. Using forceps transfer the slides from TBS into the Blocking solution. Leave for 30 minutes to 1 hour.
• Make a humid chamber: Using an opaque container, put whatman paper in the bottom and wet with TBS. Put in broken 1 ml pipettes to put the slides on to keep them off the paper.

Mix together (triterate):

• Blocking solution
• primary antibody #1
• primary antibody #2 (if double labelling)

 
• Lay out coverslips on counter.
• Put 20 ul of dilution of primary antibody on each coverslip.
• Tap dry a slide (there will still be a lot of liquid on the slide)
• Touch the slide to the coverslip.
• Put in humid chamber.
• Leave at room temperature for 1 hour.

• Dip slide in TBS to remove coverslip.
• Put in TBS for 5-10 minutes.
• Put in TBS for 5-10 minutes.

Mix together (triterate):

• Blocking solution
• secondary antibody #1
• secondary antibody #2 (if double labeling)

 
• Lay out coverslips on counter.
• Put 20 ul of secondary antibody on each coverslip.
• Tap dry a slide
• Touch the slide to the coverslip.
• Put in humid chamber.
• Leave at room temperature for 1 hour.

• Dip slide in TBS to remove coverslip.
• Put in TBS for 10-20 minutes. (Can add Dapi to this rinse, if you want, and if you are not using FITC)
• Put in TBS for 10-20 minutes.

• Lay out coverslips on counter. Check for dust etc.
• Put about 20 ul of Glycerol with 2.5%n-propylgallate on each coverslip.
• Hongbing had a note that she used 100 mM Tris-Cl pH 8.5, 80% glycerol, 2% n-propylgallate.
• Tap slide on some paper towels. Wipe back side of slide on a kimwipe. Tap off excess moisture. Pick up a coverslip using the slide. Put facedown on a kimwipe. With another kimwipe press on the back side of the slide, remove as many air bubbles as possible. Tack down the coverslip with a dab of nail polish (not necessary). Put in a cardboard holder at -20^oC.