Make fresh daily:

• 100 ul 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

Salivary gland dissection buffer (same thing as we use for permanganate footprinting: 130 mM NaCl, 5 mM KCl, 1.5 mM CaCl₂)

Preparing the glands.

1. Place 2 to 3 pairs of glands, dissected in salivary gland dissection buffer, into a drop of salivary gland dissection buffer on the surface of the silicon disk.
2. Carefully pipette away the dissection buffer and cover the glands with 50 ul of Solution A. (Use late 3rd instar larvae from a healthy, uncrowded bottle.)
3. After 5 to 10 seconds, pipette away Solution A and then cover the glands with 50 ul of solution B for 30 seconds.  (This is the fixation step and the time can be varied to obtain optimal results)
4. Pipette away solution B and cover the glands with 50 ul of solution G.
5. Put 10 ul of solution G on a siliconized cover slip placed at the edge of the bench.  Scoop up the glands with a tweezer prong and transfer the glands to the drop on the cover slip.
Siliconize the cover slip by placing a drop of Sigmacote on the cover slip and spreading it around with a Kimwipe.  Wash the cover slip with water and then with ethanol.
6. Let the glands set for 3 minutes.
7. Place a slide on the cover slip, approaching the coverslip from an angle.  Target the coverslip so that its edge is about 0.5 cm from the end of the slide opposite the frosted end.  Spread the chromosomes by first tapping on coverslip directly over glands with a needle probe. Its good for the coverslip to move around about 1 to 2 millimeters from its original location.  Check the the slides after each period of tapping to see if the chromosomes have spread sufficiently.  Continue with the tapping and looking until the glands have spread or until the solution has evaporated enough so the coverslip is no longer sliding around when it is tapped.
8. Flip the slide over and place it on a paper towel on the bench top.  With a kimwipe between your fingers and the slide, use one hand to hold the slide in place and the thumb of your other hand to press down on the slide over the cover slip.  Press firmly for a few seconds.  It is important to keep prevent the slide from moving horizontally as the coverslip may move and smear the chromosomes.  The glands should look flattened.
9. Flip the slide right side up, and put the kimwipe between your fingers and the surface of the one edge of the coverslip.  Hold the coverslip in place as you gently scratch over the surface of the coverslip with the needle probe.  This helps tack down the chromosomes to the surface of the slide.
10. Very carefully (using forceps) put the slide into liquid nitrogen (lower them in very slowly or they will crack), making sure the slide is completely covered! Leave for 3 to 5 minutes.
I like to start the next squash while this slide is in the liquid nitrogen.  While the glands for the next slide are being fixed in the drop of solution G, I remove the slide that is in the liquid nitrogen.
11. Recover the slide from the liquid nitrogen with forceps, blow on the coverslip so it becomes frosty and flick off coverslip with a razor blade.  Quickly mark where coverslip was with a diamond scribe. Then put slide into coplin jar with 95% EtOH (EtOH should cover slide).
12. If storing slides overnight, put coplin jar of 95% EtOH and slides into refrigerator.
13. Store them for 24 hrs maximum.

Staining the chromosomes for immunofluorescence analysis.

For 10 slides, you'll need 1 liter of TBS: 150 mM NaCl, 10 mM Tris-Cl pH 7.0-7.5
Prepare a humid chamber. Line the bottom of a plastic container with wet paper towels and place small petri dishes on the towels to provide a surface for supporting the slides.
Obtain one standard petri dish for each slide - these can be reused after they have been rinsed with water.

1. Rehydrating Samples

• Transfer the slides from EtOH to a coplin jar containing TBS. Leave for 5-10 minutes.
• Transfer the slides to another coplin jar containing fresh TBS.  Leave for 5-10 minutes.

• The blocking solution is 10% Fetal Bovine Serum (with 0.05% Sodium Azide) in TBS. Place a 20 ul drop of blocking solution on a coverslip. Remove a slide from the coplin jar and allow excess TBS to drain off.  With a Kimwipe, wipe excess TBS from the areas surrounding the area of the glands.  Place the slide on the coverslip so the blocking solution covers the glands.  Leave for 30 to 60 minutes in a humid chamber.
• Place one petri dish per slide on the bench so that one side of the dish is resting on the petri dish lid - as a result, the dishes are resting at an angle.  Put 20 ml of TBS in each dish.  Place the slide with the coverslip down in the dish so that the end of the slide opposite the coverslip straddles the edge of the dish.  This should submerge the coverslip without necessarily submerging the opposite side of the slide.  The coverslip usually falls off within a minute - jiggle the slide gently if needed to release the coverslip.  Leave the slide straddling the edge of the dish while you setup to apply the primary antibody.  After applying the antibody, leave the dishes filled with TBS for use as the first wash after the antibody binding.
When its convenient, I dry the coverslip with a Kimwipe and reuse it during incubation with the secondary antibody.

Mix together (triterate):

• Blocking solution (10% FBS in TBS)
• primary antibody #1
• primary antibody #2 (if double labeling - note that the Hoechst stain for DNA can interfere with weak signals produced by green fluorescing secondaries such as fluorescene)

 
• Lay out coverslips on counter.
• Put 20 ul of dilution of primary antibody on a coverslip.
• Remove the slide from the petri dish and allow excess moisture to drain from the slide.  Wipe excess moisture from the region surrounding the original location of the coverslip.
• Touch the slide to the coverslip.
• Put in humid chamber.
• Leave at room temperature for 1 hour.

• Place slides upside down in the angled petri dishes that contain 20 ml of TBS so that the slide straddles the edge of the dish.  The coverslips should fall off.  Soak the slides for 5 to 10 minutes.
• Set up a second row of angled petri dishes that each contain 20 ml of TBS.  Transfer the slides to the second angled dish of TBS and soak for 5-10 minutes.

Mix together (triterate):

• Blocking solution
• secondary antibody #1 (secondaries are usually diluted 1:100)
• secondary antibody #2 (if double labeling)

 
• Lay out coverslips on bench.
• Put 20 ul of secondary antibody on each coverslip.
• Remove excess moisture from a slide.
• Touch the slide to the coverslip.
• Put in humid chamber.
• Leave at room temperature for 1 hour.

• Place slides upside down in the angled petri dishes that contain 20 ml of TBS so that the slide straddles the edge of the dish.  The TBS can contain a 1/100000 dilution of the hoechst stock solution used for fluorescent quantitation of DNA.  Soak the slides for 10-20 minutes.
• Transfer the slides to fresh TBS in another angled petri dish and soak for 10-20 minutes.

• Lay out coverslips on bench.
• 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.
• Wipe dry the area outside the original coverslip location on the slide.  Place the slide on the drop of glycerol solution so the coverslip covers the glands.  Air bubbles can be squeezed out from under the coverslip by pressing on the surface of the coverslip with a yellow pipette tip.  Put the slides face down on a kimwipe.  Transfer the slides to fresh kimwipes until most of the excess solution as be blotted from the edges of the coverslip.  Store the coverslips horizontally in the freezer.  Alternatively, the slides can be stored vertically by putting nail polish around the perimeter of the coverslip. Store slides at -20^oC - they last for at least a few months.